CN1922318A - Methods for nucleic acid isolation and kits using solid phase material - Google Patents

Abstract

The present invention provides methods and kits for isolating nucleic acid from a sample, preferably from a biological sample, using a microfluidic device and sedimenting reagent.

Description

Use the method and the test kit of solid phase material isolating nucleic acid

Background of invention

Nucleic acid (for example, DNA and RNA) from composite interstitial substance such as blood, tissue sample, bacterial cell substratum and medical jurisprudence sample separate and purifying in genetic research, nucleic acid probe diagnostics, medical jurisprudence DNA check with to need other field of nucleic acid amplification be important method.The known multiple method for preparing amplification procedure with nucleic acid in this area, however each method all has each its limitation.

Relate to use density gradient separation peripheral blood lymphocytes (PBMC) from the most popular method of separation of whole blood DNA.Though this method is used effectively for research, it is not suitable for conventional integrated high-throughput microfluidic device usually.

The hypotonic buffer liquid that comprises the non-ionic type sanitising agent can be used for red corpuscle (RBC) and white corpuscle (WBC) cracking and keeps nucleus intact.In another approach, when the freeze thawing of whole blood process, has only the RBC cracking.Endorsing with by centrifugal recovery of undamaged WBC or they.For the RBC cracking is not destroyed WBC, can also make the use dilution as a kind of method.Other method that is used for the selective splitting of RBC comprises uses ammonium chloride or quaternary ammonium salt, and makes RBC through the hypo-osmoticity shock in the presence of hypotonic buffer liquid.Yet, in the ordinary method of one of these methods of use, the material of inhibition PCR (as, enzyme inhibitors) and nuclear and/or nucleic acid cosedimentation.Remove these inhibitor before must in the high-throughput microfluidic device of routine, analyzing.

Though for example boil, with the protease hydrolysis, be exposed to the extraction that processing under ultrasonic wave, sanitising agent or the highly basic has been used to DNA, alkaline extraction is one of the simplest method.For example, United States Patent (USP) 5,620,852 people such as () Lin described short reach at room temperature use in time of 1 minute alkali (as, NaOH) handle and effectively extract DNA from whole blood.Yet in order to obtain clean DNA, it is necessary removing dehemoglobinize and plasma proteins.This by using of short duration water-washing step to realize, for example by blood is suspended in the water, carries out centrifugal subsequently, abandoning supernatant, then with NaOH extract bead (pellet) (referring to for example, Biotechniques, Vol.25, No.4 (1998), the 588th page).Being used for a large amount of water of lysing cell makes this method not be suitable for the microfluidic device of standard.

United States Patent (USP) 5,010,183 people such as () Kellogg have been described the centrifugal microfluid based platform that uses alkaline lysis to extract DNA from blood.This method relate to primary sample (as, the whole blood or the E.Coli suspension of 5 microlitres (μ L)) mix with 10 milli (mM) NaOH that rub of 5 μ L, be heated to 95 ℃, keep 1-2 minute so that lysis, released dna and make protein denaturation to suppress PCR, by mixing with the 16mM TRIS-HCl (pH 7.5) of 5 μ L, the split product after the neutralization is mixed thermal cycling subsequently with liquid PCR reagent and the primer of 8-10 μ L with the split product neutralization.Regrettably, though amount of reagent is little and be suitable for microfluidic device, the downstream processing of DNA is still challenge in the microfluidic device.

Another ordinary method is used the phenol chloroform extraction.Yet it need use poisonous and have corrosive chemical, and is not easy to realize automatization.

Solid phase extractions also is used to separate nucleic acid.For example, from a method of nucleic acid source isolating nucleic acid relate to reaction vessel with the suspension of silicon dioxide granule with pass through buffered chaotropic agent such as guanidine thiocyanate and mix, add sample subsequently.In the presence of chaotropic agent, nucleic acid is adsorbed on the silicon-dioxide, makes it from liquid phase separation by centrifugal, washes with alcohol-water mixture, uses rare aqueous solution of buffer agent wash-out at last.The silicon-dioxide solid phase extractions need use pure washing step to remove residual chaotropic agent wash-out nucleic acid not; Yet must extreme care be used in the later step nucleic acid is increased or the susceptibility enzyme of modification to prevent to be suppressed at the alcohol of removing all traces (thermal evaporation or wash with another kind of very volatile and inflammable solvent).Water or elution buffer wash-out nucleic acid then.This combination, rinsing and elution process are the principles of many commercial reagent box, as Qiagen (Valencia, CA); Yet this method bothers and comprises a plurality of washing steps very much, makes it be difficult to be applicable to microfluidic device.

Ion exchange method produces high-quality nucleic acid.Yet ion exchange method causes the existence of high-caliber salt, must be removed before nucleic acid can be used further.

International open WO 01/37291 A1 (MagNA Pure) described the application of magnetic glass particle and wherein by with the dedicated buffering liquid cultivation that comprises chaotropic salt and Proteinase K with the separation method of sample dissociation.Add the magnetic glass particle, be included in the surface that whole nucleic acid in the sample are incorporated into particle.Remove unconjugated material by the plurality of washing step.At last, at high temperature use the low salt buffer wash-out, obtain whole nucleic acid of purifying.

Also having another ordinary method to relate to is applied on the hydrophobic organic polymer solid phase biological sample optionally to hold back nucleic acid and to remove the nucleic acid that is trapped with nonionic surface active agent subsequently.Another method relates to nonionic surface active agent handles the hydrophobic organic polymer material, washing surface, and make treated SOLID ORGANIC polymer materials contact biological sample subsequently, be incorporated into the amount of the nucleic acid on the organic polymer solid phase with minimizing.Though these solid phase methods still need other method for being effective means from the biological sample isolating nucleic acid, particularly be fit to the method for microfluidic device.

Aforementioned discussion open and prior art do not constituted these data are admitted as the part of disclosed, known or common sense.

Summary of the invention

The invention provides the method that is used for isolating nucleic acid, preferred purifying and reclaims nucleic acid.

Isolating nucleic acid can be used in the test of the existence of concrete nucleic acid in the test sample for example according to the present invention.This test has importance in the prediction of disease and diagnosis, medical jurisprudence, epidemiology and public health.For example, separated DNA can be used for detecting the existence of individuality infective virus or mutator gene through hybridization and/or amplification, is used to measure the probability that this individuality suffers from the disease of communicable disease or genetics origin.Detect the infective virus in the sample in the screened hundreds of or thousands of samples or the ability of sudden change and significant importance is arranged for early diagnosis that is in the colony under the disease danger or epidemiology, for example, HIV infection, cancer or to the early detection of the susceptibility of cancer, or in neonatal general investigation of desease, wherein early detection has diagnosis of helping and treatment.In addition, method of the present invention can also be used for the fundamental research laboratory, is used for from cultured cells or biochemical reaction isolating nucleic acid.Nucleic acid can be used for enzymatically modifying such as digestion with restriction enzyme, order-checking and amplification.

The invention provides be used for from comprise nucleic acid (as, DNA, RNA, PNA) the method and the test kit of sample separation nucleic acid, described nucleic acid can be included in or be not included in and contain in the nuclear cell (as, white corpuscle).These methods relate to from inhibitor such as protoheme (heme) and degraded product thereof (as, iron ion or its salt) final isolating nucleic acid, inhibitor is undesirable, because they can the suppression of amplification reaction (as, the situation in the PCR reaction).

Certain embodiments of the present invention relate to and remain on inhibitor in the solid phase material or remain on the nucleic acid that (that is, makes inhibitor be attached to this material) on the solid phase material and do not keep significant quantity.Suitable solid phase material generally include have connected catch the position (as, huge legendary turtle is closed functional group) any form (as, particle, protofibril, form membrane) solid substrate, be applied to coating reagent (preferred surfactant) on the solid phase material or both.

In one embodiment, the invention provides from the method for sample separation nucleic acid, this method comprises: provide to comprise that the material and the inhibitor that contain nucleic acid (are generally comprised within the cell; The described material that contains nucleic acid and contain the cell of inhibitor can be identical or different) sample; If sample comprises the cell that contains inhibitor, then this method comprises and randomly makes sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor; Form sample (usually, being lysate sample) enrichment region; Wherein sample (usually, being lysate sample) enrichment region comprises material and the inhibitor that contains nucleic acid; Sample (usually, being lysate sample) enrichment region and the inferior enrichment region of sample (usually, being lysate sample) are separated basically; Make isolating sample (usually, be lysate sample) enrichment region contact solid phase material, make at least a portion inhibitor (promptly, at least a portion of at least a inhibitor) preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; Before making isolating sample concentration district contact solid phase material, simultaneously or afterwards randomly further the cracking material that contains nucleic acid be used to discharge nucleic acid; Separate with the solid phase material that has adhered at least a portion inhibitor with the material and/or the nucleic acid that make at least a portion contain nucleic acid.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: the sample that comprises the cell that contains inhibitor and contain nuclear cell (described contain the cell of inhibitor and contain nuclear cell can be identical or different) is provided; Make biological sample effectively destroy cytolemma and discharge nucleus and inhibitor and form under the condition of lysate sample and contact nonionic surface active agent; Form the lysate sample enrichment region; Wherein the sample concentration district comprises nucleus and inhibitor; The lysate sample enrichment region is separated basically with sample time enrichment region; Make isolating lysate sample enrichment region contact solid phase material make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; Before making isolating sample concentration district contact solid phase material, simultaneously or afterwards randomly further lysing cell nuclear be used to discharge nucleic acid; Separate with the solid phase material that has adhered at least a portion inhibitor with the material and/or the nucleic acid that make at least a portion contain nucleic acid.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: provide microfluidic device, the separate chamber that it comprises feed compartment, the valve treatment chamber is arranged and comprise solid phase material; The sample that comprises the material that contains nucleic acid and inhibitor (usually, be included in the cell, described contain the material of nucleic acid and contain the cell of inhibitor can be identical or different) is provided; Sample is placed feed compartment; If sample comprises the cell that contains inhibitor, then randomly sample is contacted first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor; Sample (usually, being lysate sample) has been transferred to the valve treatment chamber; Form sample (usually, being lysate sample) enrichment region in the valve treatment chamber is arranged, wherein sample (usually, being lysate sample) enrichment region comprises material and the inhibitor that contains nucleic acid; Sample (usually, being lysate sample) enrichment region is separated basically with inferior enrichment region; Isolating sample (usually, being lysate sample) enrichment region is transferred to the separate chamber and contacted with solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material; Wherein solid phase material comprises and catches position (as, chelating functional group), is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; Before isolating sample concentration district contact solid phase material, simultaneously or afterwards randomly further cracking contain the material of nucleic acid to discharge nucleic acid; Separate with the solid phase material that has adhered at least a portion inhibitor with the material and/or the nucleic acid that make at least a portion contain nucleic acid.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: provide microfluidic device, the separate chamber that it comprises feed compartment, the valve treatment chamber is arranged and comprise solid phase material; The sample that comprises the cell that contains inhibitor and contain nuclear cell (described contain the cell of inhibitor and contain nuclear cell can be identical or different) is provided; Sample is placed feed compartment; Make sample effectively destroy cytolemma and discharge nucleus and inhibitor and form under the condition of lysate sample and contact nonionic surface active agent; Lysate sample has been transferred to the valve treatment chamber; Form the lysate sample enrichment region in the valve treatment chamber is arranged, wherein the lysate sample enrichment region comprises nucleus and inhibitor; The lysate sample enrichment region is separated basically with inferior enrichment region; Isolating lysate sample enrichment region is transferred to the separate chamber contact, make at least a portion inhibitor preferentially be attached to solid phase material with solid phase material; Wherein solid phase material comprises and catches position (as, chelating functional group), is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; Before isolating sample concentration district contact solid phase material, simultaneously or afterwards randomly further lysing cell nuclear to discharge nucleic acid; With make at least a portion nucleus/or nucleic acid separate with the solid phase material that has adhered at least a portion inhibitor.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: the sample that comprises nucleic acid and inhibitor is provided; Make sample contact solid phase material make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; If before making lysate sample contact solid phase material, simultaneously or afterwards randomly the material that contains nucleic acid that exists of cracking to discharge nucleic acid; With separate with the solid phase material that has adhered at least a portion inhibitor by material and/or the nucleic acid that makes at least a portion contain nucleic acid with the eluent wash-out, condition is that eluent is not a nonionic surface active agent.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: provide to comprise the material that contains nucleic acid (as, nucleus) and inhibitor (usually, be included in the cell, the described material that contains nucleic acid and contain the cell of inhibitor can be identical or different) sample; If sample comprises the cell that contains inhibitor, randomly make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor; Make sample (usually, be lysate sample) the contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination (preferably, being tensio-active agent) thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; Before making lysate sample contact solid phase material, simultaneously or afterwards randomly further cracking contain the material of nucleic acid to discharge nucleic acid; With separate with the solid phase material that has adhered at least a portion inhibitor by material and/or the nucleic acid that makes at least a portion contain nucleic acid with the eluent wash-out, condition is that eluent is not a nonionic surface active agent.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: the sample that comprises the material that contains nucleic acid (as, nucleus) and contain the cell (described cell can be identical or different) of inhibitor is provided; Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor; Make lysate sample contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination (preferably, being tensio-active agent) thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; The material that makes at least a portion contain nucleic acid separates with the solid phase material that has adhered at least a portion inhibitor; With after separating the material that contains nucleic acid, further cracking contains the material of nucleic acid to discharge nucleic acid.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: provide microfluidic device, the separate chamber that it comprises feed compartment and comprises solid phase material; Provide and comprise that the material and the inhibitor that contain nucleic acid (are included in the cell usually; The described material that contains nucleic acid and contain the cell of inhibitor can be identical or different) sample; Sample is placed feed compartment; If sample comprises the cell that contains inhibitor, then randomly make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor; Sample (usually, being lysate sample) is transferred to the separate chamber to contact solid phase material and to make at least a portion inhibitor preferentially be attached to solid phase material; Before making sample contact solid phase material, simultaneously or afterwards randomly further cracking contain the material of nucleic acid to discharge nucleic acid; Wherein solid phase material comprises and catches position (as, chelating functional group), is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; With separate with the solid phase material that has adhered at least a portion inhibitor by material and/or the nucleic acid that makes at least a portion contain nucleic acid with the eluent wash-out, condition is that eluent is not a nonionic surface active agent.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: provide microfluidic device, the separate chamber that it comprises feed compartment and comprises solid phase material; The sample that comprises material that contains nucleic acid and the cell that contains inhibitor (they can be identical or different) is provided; Sample is placed feed compartment; Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor; Lysate sample is transferred to the separate chamber to contact solid phase material and to make at least a portion inhibitor preferentially be attached to solid phase material; Wherein solid phase material comprises and catches position (as, chelating functional group), is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; Separate with the solid phase material that has adhered at least a portion inhibitor with the material that makes at least a portion contain nucleic acid; With after separating the material that contains nucleic acid, further cracking contains the material of nucleic acid to discharge nucleic acid.

The present invention also is provided for implementing the test kit of the whole bag of tricks of the present invention.

Definition

" nucleic acid " should have implication as known in the art, and be meant DNA (as, genomic dna, cDNA or plasmid DNA), RNA (as, mRNA, tRNA or rRNA) and PNA.It can be various ways, includes but not limited to bifilar or sub-thread structure, ring-type, plasmid, short relatively oligonucleotide, peptide nucleic acid(PNA) (being also referred to as PNA) (as people such as Nielsen, Chem.Soc.Rev. is 26, described in the 73-78 (1997)), or the like.Nucleic acid can be genomic dna, and it can comprise a complete karyomit(e) or a chromosomal part.DNA can comprise encoding sequence (as, mRNA, tRNA and/or rRNA are used to encode) and/or non-coding sequence (as, kinetochore, telomere, intergenic region, intron, transposon and/or microsatellite sequence).Nucleic acid can comprise any naturally occurring Nucleotide and carry out the Nucleotide of artificial or chemically modified, sudden change Nucleotide etc.Nucleic acid can comprise non-nucleic acid component, as peptide (as in PNA), marker (radio isotope or fluorescence labelling thing), or the like.

" material that contains nucleic acid " is meant the source of nucleic acid, as cell (as, the red corpuscle of white corpuscle, stoning), nucleus or virus or contain the structure that contains nucleic acid any other composition (as, plasmid, clay or viroid, archeobacteria).Cell can be protokaryon (as, gram-positive microorganism or Gram-negative bacteria) or eucaryon (as, hemocyte or histocyte).Be virus if contain the material of nucleic acid, it can comprise RNA or DNA genome; It can be fatal, attenuation or noninfectious; And it can infect protokaryon or eukaryotic cell.That the material that contains nucleic acid can be is naturally occurring, through manually modified or produce through artificial.

" isolating " is meant the isolated nucleic acid of at least a portion inhibitor from sample (that is at least a portion of at least a inhibitor) the material of nucleic acid (or contain).This comprises from other material such as cellular component such as protein, lipid, salt and other inhibitor isolates required nucleic acid.More preferably, isolating nucleic acid is purifying basically." purifying basically " is meant separation at least 3 piks/microlitre (pg/ μ L), preferred at least 2 nanograms/microlitre (ng/ μ L), more preferably the nucleic acid of 15ng/ μ L at least makes the amount of inhibitor in the primary sample reduce at least 20% simultaneously, preferred reduction at least 80% more preferably reduces at least 99%.Impurity is cellular component and the nuclear components the solvent in sample normally, as protoheme and associated products (teichmann's crystals, protoheme) and metal ion, protein, lipid, salt etc.Therefore, term " purifying basically " generally be meant from sample separation go out most of inhibitor (as, protoheme and degraded product thereof), make and remove the compound of using subsequently that can hinder isolating nucleic acid at least in part.

" be attached to " or " adhering to " or " combination " is that reversibility by number of mechanisms is detained, described mechanism comprises more weak power, as Van der Waals interaction, electrostatic interaction, affinity in conjunction with or physics hold back.The use of this term does not hint mechanism of action, and comprises absorption mechanism and mechanism of absorption.

" solid phase material " is meant inorganic or organic materials, the preferred polymkeric substance of making by repeating unit, and repeating unit can be identical or different, is the compound in organic and/or inorganic, natural or synthetic source.This comprise homopolymer and heteropolymer (as, copolymer, terpolymer, tetrapolymer etc., it can be for example random or block).This term comprises the polymkeric substance of fibrous or particle form, and it can easily prepare by means commonly known in the art.This material forms porous matrix usually, though for some embodiment, solid phase also refers to solid surface, as the atresia sheet material of polymer materials.

Solid phase material can comprise catches the position." catch the position " and be meant the position that is used for attachment material on the solid phase material.Usually, catch the position comprise and be covalently attached to or otherwise be connected in (as, hydrophobicity is connected in) functional group or molecule on the solid phase material.

Phrase " is applied to the coating reagent on the solid phase material " and is meant the material at least a portion that is coated on solid phase material, as, be coated on the material at least a portion of fibril-matrix and/or adsorptivity particle.

" tensio-active agent " is meant that reducing it is dissolved in the material of the surface tension or the interfacial tension of medium wherein.

" highly basic " is meant complete dissociated alkali in water, as, NaOH.

" polyelectrolyte " is meant the ionogen as electropolymer, and it has usually than higher molecular weight, as polystyrolsulfon acid.

" the polymkeric substance barrier of alternative infiltration " is meant the polymkeric substance barrier according to size and the alternative transhipment of electric charge liquid.

" enrichment region " is meant the bead form that can be, and has the sample area of the material that contains nucleic acid, nucleus and/or the nucleic acid of higher concentration with respect to inferior enrichment region.

As used in this article, " separate basically ", particularly in sample concentration district and the sample time isolating situation of enrichment region, be meant remove the sample total amount be lower than at least 40% of 25% nucleic acid (no matter it is for free or in nucleus or in other contains the material of nucleic acid) total amount.Preferably, be lower than at least 75% of 10% nucleic acid total amount from what the rest part of sample was isolated the sample total amount.More preferably, be lower than at least 95% of 5% nucleic acid total amount from what the rest part of sample was isolated the sample total amount.

" inhibitor " is meant and is used for for example enzyme inhibitors of amplified reaction.The example of this inhibitor generally includes iron ion or its salt (as, Fe ²⁺Or its salt) and other metal-salt (as, alkalimetal ion, transition metal ion).Other inhibitor can comprise protein, peptide, lipid, carbohydrate, protoheme and degraded product thereof, urea, bile acide, humic acid, polysaccharide, cytolemma and kytoplasm component.The main inhibitor of PCR in human blood is oxyphorase, lactoferrin and IgG, and it is present in respectively in red corpuscle, white corpuscle and the blood plasma.Method of the present invention makes at least a portion inhibitor (that is at least a portion of at least a inhibitor) and the separating substances that contains nucleic acid.As discussing herein, contain inhibitor cell can with contain nuclear cell or other material that contains nucleic acid is identical.Inhibitor can be included in the cell or can be positioned at the extracellular.The extracellular inhibitor comprises that all are not included in intracellular inhibitor, and it comprises those inhibitor that for example are present in serum or the virus.

The inhibitor that " preferentially makes at least a portion inhibitor be attached to solid phase material " to be meant one or more types than the material that contains nucleic acid (as, nucleus) and/or nucleic acid be attached on the optional solid phase material with bigger degree, and usually the material that contains nucleic acid and/or the nucleus of signal portion are attached on the solid phase material.

" microfluid " is meant the device with one or more fluid channels, chamber or conduit, its at least one inner cross-sectional dimension that has (as, the degree of depth, width, length, diameter etc.) less than 500 μ m, be generally 0.1 μ m to 500 μ m.In the device that the present invention uses, at least one cross-sectional dimension of preferred microscale channel or chamber be 0.1 μ m to 200 μ m, more preferably 0.1 μ m to 100 μ m, be generally 1 μ m to 20 μ m.Usually, microfluidic device comprises a plurality of chambers (treatment chamber, separate chamber, mixing section, waste chamber, thinner chamber, amplified reaction chamber, feed compartment etc.), and each chamber is defined for the capacity that comprises sample; And there is at least one to distribute passage to connect a plurality of chambers of array; For example, wherein at least one chamber in the array can comprise that at least one treatment chamber in solid phase material (thereby it often is known as the separate chamber) and/or the array can comprise lytic reagent (thereby it often is known as mixing section).

Term " comprises " and variant is not to have restrictive sense when these terms appear in teachings and the claim.

As used in this article, " one ", " one ", " being somebody's turn to do ", " at least one " and " one or more " are used interchangeably and are meant one or more.

Also in this article, the narration of the numerical range of representing by end points comprise all numbers of being comprised in this scope (as, 1 to 5 comprises 1,1.5,2,2.75,3,3.80,4,5 etc.).

Foregoing invention content of the present invention is not used in describes each disclosed embodiment or all execution of the present invention.Following description more specifically illustrates exemplary embodiment.Several places in the application provide guidance by enumerating of embodiment, and described embodiment can use by various combination.In each case, it is just representational to enumerate example, it should be interpreted as the exclusive example of enumerating.In addition, described different embodiments, wherein a plurality of elements of each embodiment can be used to other embodiment, even without describing particularly.

Description of drawings

Fig. 1-2 represents to be used for the microfluidic device of some method of the present invention.

The detailed description of exemplary

The invention provides and be used for, biological sample normally, isolating nucleic acid, the preferred several different methods and the test kit of the nucleic acid of pure form basically from sample.The invention provides be used for from comprise nucleic acid (as, DNA, RNA, PNA) the method and the test kit of sample separation nucleic acid, described nucleic acid can be included in or be not included in and contain in the nuclear cell (as, white corpuscle).

Should be appreciated that though this method relates to from sample separation nucleic acid, this method may not be to remove nucleic acid from the material that contains nucleic acid (as, nucleus).That is to say, may need other step, with further from for example nucleus isolating nucleic acid.

Method of the present invention relates to finally makes nucleic acid and inhibitor such as protoheme and degraded product thereof (as, molysite) separate, and inhibitor is undesirable, but because the reaction of their suppression of amplification (as, the situation in the PCR reaction).More specifically, method of the present invention relates at least a portion nucleic acid that makes in the sample and separates with at least a portion of at least a inhibitor.Preferable methods relates to all basically inhibitor of removing in the sample that comprises nucleic acid, makes that nucleic acid is pure basically.For example, the ultimate density of ferruginous inhibitor is not more than about 0.8 micromole (μ M), and it is the permissible level that exists in the conventional PCR system.

In order to obtain clean DNA from whole blood, expectation removes dehemoglobinize and plasma proteins usually.When erythrocyte splitting, discharge protoheme and related compound, they suppress the Taq polysaccharase.Normal hemoglobin concentration is per 100 milliliters (mL) 15 grams (g) in the whole blood, based on this, is about 10 millis rub (mM) through the concentration of the protoheme in the hemolytic whole blood.For PCR is carried out satisfactorily, the concentration of protoheme should be reduced to micromole (μ M) level.This can realize by dilution, or for example remove inhibitor with inhibitor bonded material by use and realize.

Usually, the sample that will comprise nucleic acid is handled in circulation (flow-through) container, though this container is not essential requirement of the present invention.Preferably, for some method of the present invention, treatment facility is a microfluidic structures.

Sample

Method of the present invention can be used for from various samples, biological sample particularly, isolating nucleic acid, described sample such as body fluid (as, whole blood, serum, urine, saliva, cerebrospinal fluid, seminal fluid or synovia lymph liquid), different tissues (as, skin, hair, tongue fur, ight soil, knurl or organ such as liver or spleen), cell culture or cell culture supernatant liquid etc.Sample can be foodstuff samples, drink sample, fermenting broth, the diagnosis that is used for disease or illness or disposal or monitoring or treatment clinical sample, medical jurisprudence sample, agriculture sample (as, derive from plant or animal) or environmental sample (as, soil, dirt or rubbish).

Biological sample is to have biogenetic derivation or biochemical those of originating.Those biological samples that are suitable in the inventive method can be from Mammals, plant, bacterium or yeast source.Biological sample can be independent cells form or is organizational form.Cell or tissue can be from isolated culture.Importantly be, certain embodiments of the present invention use without any pre-treatment (as, cracking, filtration etc.) whole blood as interested sample.

For some embodiment, sample such as whole blood can carry out pre-treatment by centrifugal, and from blood separation white corpuscle (that is buffy coat) and as the sample in the inventive method.

For some embodiment, sample can experience ultracentrifugation with concentrating sample before it experiences processing of the present invention.

Sample can be dissolving or is dispersed in solid sample in water or the organic medium (as, solid tissue), or therefrom with the solid sample of nucleic acid extraction in water or the organic medium.For example, sample can be organ homogenate (as, liver, spleen).Therefore, sample can comprise the nucleic acid (if particularly it is solid sample) through extracting in advance.

The type of sample is not restriction of the present invention.Yet, usually, sample comprise the material that contains nucleic acid and need with the inhibitor of separate nucleic acid.In this context, the material that contains nucleic acid be meant various cells (as, white corpuscle, bacterial cell), nucleus, virus or contain the structure that contains nucleic acid any other composition (as, plasmid, clay or viroid, archeobacteria).In some preferred embodiment of this method, the material that contains nucleic acid comprises nucleus.In certain embodiments, when contacting with solid phase material as herein described, this nucleus is undamaged (, uncracked basically).

In certain embodiments, sample can be the part cracked (as, pre-cracking is with release inhibitor), in this case, method of the present invention may need cracking or cracking fully.Therefore, that sample can comprise is free (as, not in cell) nucleic acid and free (as, not in cell) inhibitor.

Isolating (promptly, isolating with inhibitor) nucleic acid can be used for (preferably without being further purified or washing) multiple application (as, amplification, order-checking, mark, cancellation, digestion with restriction enzyme, connection, reversed transcriptive enzyme, hybridization, southern blotting technique, RNA trace etc.).Particularly, it can be used for measuring the genome of main body.It can be used for diagnosing microorganism in the sample (as, bacterium, virus) existence, and can be used for monitoring and/or treating the damage that causes by microorganism subsequently to sample source.Method of the present invention, material, system and test kit be particularly suitable for preparation high-throughput or automatization handle use in (particularly microfluid system) amplification technique (as, PCR, LCR, MASBA, SDA and bDNA) use nucleic acid extractive.Therefore, for certain embodiments of the present invention, isolating nucleic acid is transferred in the amplified reaction chamber (as the PCR sample chamber in the microfluidic device).

Can be according to the present invention from impure, partial-purified or pure sample separation nucleic acid (that is, separating) with inhibitor.The purity of primary sample is not vital because can from addition very impure sample separation go out nucleic acid.For example, can obtain nucleic acid from impure biologicfluid sample such as blood, saliva or tissue.If the primary sample of expectation higher degree, can make sample before experience method of the present invention according to well known to a person skilled in the art that any ordinary method handles.For example, can handle sample, make and before sample experiences method of the present invention, remove some impurity, as insoluble substance.

Isolating nucleic acid as described herein can have any molecular weight and be sub-thread form, bifilar form, annular, plasmid etc.Can multiple nucleic acid is separated from one another (as, from the DNA isolation of RNA, or separate double-stranded DNA from single stranded DNA).For example, can use method of the present invention to separate to have about 10 little oligonucleotide or nucleic acid molecule, have about 1000 bases to the longer molecule of about 10,000 base length even have the macromolecule nucleic acid that about 50kb arrives about 500kb to about 50 base length.In certain aspects, isolating nucleic acid can preferably have about 10 bases to about 100,000 bases according to the present invention.

The sample that contains nucleic acid can have different capacity.For example, the capacity of use can be greatly to 1 liter or little of 1 μ L or littler.The size of sample depends on the equipment that is used to implement this method usually.For microfluidic structures, it has very little capacity usually, as preferred 10 μ L (preferably being not more than 100 μ L).Should be appreciated that,, can use more jumbo sample if concentrate as passing through through pre-treatment.

For the low copy number gene, need bigger sample capacity to be present in the sample usually to guarantee interested sequence.Yet bigger sample size has more substantial inhibitor, and makes usually and himself be unsuitable for microfluidic structures.Therefore, for the situation of low copy number, may need to use 100 μ L or bigger capacity, to obtain reproducible result; Yet because higher sample capacity, the handled sample number of each microfluidic device may reduce.

In the method for the invention, being used for the concentrated centrifugation step that contains the material of nucleic acid is useful for the low copy number sample.Yet though nucleic acid concentration significantly increases in bottom of treatment chamber, inhibitor concentration is still higher.Though in supernatant liquor, remove most of inhibitor, promptly the protein among serum and the ruined RBC (as, protoheme and protoheme associated products), the enrichment region that contains nucleic acid of sample still has the inhibitor of significant quantity to exist; Yet nucleic acid is very high with the ratio of inhibitor, produces the spissated relatively sample of nucleic acid.Can make the optional solid phase material of this sample concentration district contact, as described herein, to remove residual inhibitor.

For the high copy number gene, can use the sample capacity that reaches 2 μ L for a short time, but be to use more large vol when (as, 20 μ L) circulation ratio better.In situation, can obtain higher throughput (that is the sample number of each microfluidic device processing) than low capacity.In the comparatively large capacity situation of (as, 20 μ L), may need not experience the pre-rotation step that is used for concentrating the cell that contains nucleic acid.

For some embodiment, the sample that contains nucleic acid that is applied on the solid phase material can be any amount, and this amount is by the amount decision of solid phase material.Preferably, the amount that is applied to the sample amplifying nucleic acid on the solid phase material is lower than the dry weight of solid phase material, is generally about 1/10,000 to about 1/100 (weight, nucleic acid/solid phase).The amount that is applied to the sample amplifying nucleic acid on the solid phase material can for example reach 100 grams or low 1 pik that reaches.

Preferably from the isolating required nucleic acid of the inventive method for the sample that applies at first all at least 20% of the amount of nucleic acid, more preferably at least 30%, more preferably at least 70%, most preferably at least 90%.Therefore, some preferred method of the present invention provides with high-recovery and reclaims required nucleic acid from sample.In addition, can reclaim the nucleic acid molecule of minute quantity quantitatively according to the present invention.The rate of recovery or yield depend primarily on the quality rather than the method itself of sample.Do not need from the spissated nucleic acids for preparation method of large vol because certain embodiments of the present invention provide, so the present invention has avoided the danger of nucleic acid loss.

Having too many DNA in the PCR sample may be harmful to the amplification of DNA, because there is too many mistake to draw position (misprimed site).The non-target sequence that this produces many linearities or increases by exponential manner.Because the specificity of amplification disappears along with the increase of non-target dna amount, the index with any significance degree generation target sequence does not gather.Therefore, wish the amount that control enters the DNA in each PCR sample.The amount of DNA is no more than 1 microgram/reaction usually, is generally at least 1 pik/reaction.Typical final DNA concentration is that 0.15 nanogram/microlitre is to 1.5 nanograms/microlitre in the PCR mixture.In the situation of microfluidic device, can make each sample have an amount of DNA with the sample shunting after purifying, before the PCR.Perhaps, sample sufficiently can be diluted in the sample processing device that comprises the treatment chamber that variable valve is housed as more specifically described as the following (being specially microfluidic device), make to have an amount of DNA in each PCR mixture.In diagnostic device, because leukocytic amount can be significantly different, the amount of the DNA that very difficult ex ante forecasting will be separated.Yet useful range is that per 200 μ L blood contain the DNA of 3 micrograms (μ g) to 12 μ g.For buffy coat, useful range is that per 200 μ L buffy coats contain the DNA of 25 μ g to 50 μ g.

Lytic reagent and cracking condition

For certain embodiments of the present invention, some time points in treating processes are with the lysis in the sample, particularly contain nucleic acid cell (as, white corpuscle, bacterial cell, virocyte), with the content that discharges cell and form sample (that is split product).The physics of the film that is cracked into each cell of this paper breaks, be meant the okioplast film and when having nuclear membrane the physics of nuclear membrane break.This can use standard technique to carry out, as make the hot deactivation of proteolytic enzyme subsequently by protease hydrolysis; With tensio-active agent (as, nonionic surface active agent or sodium lauryl sulphate), guanidinesalt or highly basic (as, NaOH) handle; Physical damage (as, use ultrasonic wave); Boil; Or heating/cooling (as, be heated to minimum 55 ℃ (usually to 95 ℃) and cool to room temperature or following (to 8 ℃)), it can comprise the freeze/thaw processing.Usually, if use lytic reagent, it is in the water medium, though if can be with an organic solvent during expectation.

(RBC) cracking of red corpuscle in the whole blood can not destroyed white corpuscle (WBC), be used for release inhibitor as lytic reagent by making water (that is moisture dilution).Perhaps, also can use ammonium chloride or quaternary ammonium salt to destroy RBC.Can also use hypotonic buffer liquid by hypo-osmoticity shock cracking RBC.Can pass through complete WBC of for example centrifugal recovery or their nucleus.

Usually, can use stronger lytic reagent such as tensio-active agent to be used for cracking RBC and contain nucleic acid cell (as, white corpuscle (WBC), bacterial cell, virocyte), with release inhibitor, nucleus and/or nucleic acid.For example, can use nonionic surface active agent cracking RBC and WBC, and not destroy nucleus.Can use nonionic surface active agent, cats product, anion surfactant and zwitterionics lysing cell.Useful especially is nonionic surface active agent.If expectation can be used the combination of tensio-active agent.Nonionic surface active agent such as TRITON X-100 can be joined in the TRIS damping fluid that contains sucrose and magnesium salts, be used for nuclear separation.

The amount that is used for the cracked tensio-active agent is enough high, with effective lysate sample; Also enough low, for example precipitate avoiding.The surfactant concentrations that is used for cracking process is generally at least 0.1 weight % based on the gross weight of sample.The surfactant concentrations that is used for cracking process is not more than 4.0 weight % usually based on the gross weight of sample, preferably is not more than 1.0 weight %.Usually optimize this concentration to reach complete cracking in the shortest as far as possible time, the mixture that obtains is that PCR is compatible.In fact, the nucleic acid that joins in the preparation in the PCR cocktail should seldom suppress or suppress hardly PCR in real time.

If expectation can mix use with damping fluid with tensio-active agent.Usually, sort buffer liquid provides pH to be at least 7, is up to 9 sample usually.

Usually, can use even stronger lytic reagent,, be included in any nucleus in the cell (as white corpuscle) that contains nucleic acid with cracking as highly basic.For example, United States Patent (USP) 5,620, the method that 852 people such as () Lin describe can be suitable for some method of the present invention, its relate at room temperature use alkaline purification (as, NaOH) extract DNA from whole blood short reaching in time of 1 minute.Usually, have multiple highly basic to can be used in the alkaline lysis to produce effective pH (as, 8-13, preferred 13).Highly basic is generally oxyhydroxide, as NaOH, LiOH, KOH; Has the quaternary nitrogen containing positively charged ion oxyhydroxide of (as, quaternary ammonium); And alkali such as tertiary amine, secondary amine or primary amine.Usually, alkaline concentration is at least 0.01 equivalent concentration (N), and is up to 1N usually.Usually, then with the mixture neutralization, if particularly nucleic acid experiences PCR.In another approach, can after with alkaline lysis, heat, subsequently sample be neutralized further to make protein denaturation.

Can also under heating, use Proteinase K, under higher temperature, make the hot deactivation of Proteinase K subsequently, be used for from nucleus or WBC isolating nucleic acid.

Can also use commercially available lytic reagent and neutralizing agent, as Extract-N-Amp Blood PCR test kit according to the Sigma of microfluid dimensions scale downward.(Oslo, POWERLYSE Norway) are used to make be difficult to cracked bacterium such as cracking such as staphylococcus, suis, to help some method of the present invention as deriving from GenPoint can to use stronger cracked solution.

In another approach, can use boiling method lysing cell and nucleus, released dna, and precipitate oxyphorase simultaneously.DNA in the supernatant liquor need not enrichment step can be directly used in PCR, makes this method can be used for the low copy number sample.

For infectious diseases, may need to analyze the bacterium or the virus that derive from whole blood.For example, in the situation of bacterium, white corpuscle may exist with bacterial cell bonded form.In the method for using microfluidic device, possible splitting erythrocyte is further going out bacterial cell and white corpuscle by for example centrifugation before the cracking then with release inhibitor.The concentrating part of this cell that contains nucleic acid (bacterium with leukocytic cell/nucleus) can further move in the chamber that comprises solid phase material, is used to remove inhibitor.Then, can be with for example bacteria cell cracking.

Can use heat to realize the cracking of bacterial cell.Perhaps, can use enzyme urge method (as, N,O-Diacetylmuramidase, mutanolysin) or chemical method carry out the cracking of bacterial cell.The preferred bacterium cell carries out cracking by alkaline lysis.

Using bacterium to be used for breeding plasmid is common in Study on Genome, analyzing molecules biology, preparation molecular biology etc.In the situation of the bacterium that comprises plasmid, there is the genetic material that derives from bacterium and plasmid.Can use method of the present invention to carry out purification operations, be used for from genomic dna isolated cell protein and cell debris.The supernatant liquor that comprises plasmid DNA that so obtains is called " clarifying lysate ".Can use several different methods that cleared lysate is further purified, as anion-exchange chromatography, gel-filtration or with the alcohol precipitation.

In the object lesson of the scheme that can be bonded to the bacterial cultures in the microfluidic device, the E.Coli cell culture is centrifugal and be resuspended in the TE damping fluid (10mM TRIS, 1mMEDTA, pH 7.5) and by adding 0.1M NaOH/1%SDS (sodium lauryl sulphate) cracking.Stop lysis by 3M (three moles) potassium acetate (pH 4.8) that adds 1 volume, and supernatant liquor is centrifugal.Product of cell lysis is further purified, obtains clean plasmid DNA.

Blood plasma and serum have been represented and have been submitted the most of samples that comprise virus that are used for the molecule check to.After whole blood carried out classification, blood plasma or serum sample can be used for extracting virus (that is virus particle).For example, for from viral DNA isolation, in the microfluid situation, might be at first by centrifugal blood be told serum.Use can be injected into independent serum in another chamber at the following variable valve of more specifically describing.Then can serum is centrifugal with concentrating virus, perhaps can after removing inhibitor, for example described solid phase material of use be directly used in cleavage step subsequently herein.Solid phase material can absorbent solution, makes virus particle not pass through material.Then can be with little elution volume wash-out virus particle.By heating or by enzymatic or chemical process with virolysis (for example carrying out cracking) and be used for application such as the PCR or the PCR in real time in downstream by the use tensio-active agent.Need therein in the situation of viral RNA, may need ribonuclease inhibitor is joined in the solution, to stop the RNA degraded.

Solid phase material

For certain embodiments of the present invention, have been found that inhibitor is attached to solid phase (preferred polymers) material, this material comprise any form (as, particle, protofibril, form membrane) solid substrate, preferred its has connectedly catches position (as, chelating functional group), is applied to the coating reagent (preferred surfactant) of (being its at least a portion) on the solid phase material or both.Coating reagent can be positively charged ion, negatively charged ion, nonionic or amphoteric ionic surfactant.Perhaps, coating reagent can be the polymkeric substance barrier of polyelectrolyte, highly basic or alternative infiltration.If expect, can use the various combination of coating reagent.

The solid phase material that can be used for method of the present invention can comprise the multiple organic and/or inorganic materials that for example keeps inhibitor such as protoheme and hemachrome degradation product (particularly iron ion).This material with catch position (preferred chelation group) functionalized, scribble one or more coating reagents (as, tensio-active agent, polyelectrolyte or highly basic) or both.Usually, solid phase material comprises organic polymer matrix.

Usually suitable material is chemically inert, physics and chemically stable and compatible with multiple biological sample.The example of solid phase material comprise silicon-dioxide, zirconium white, alumina bead, metallic colloid as gold, for example by the sulfydryl chemistry carry out functionalized processing be used to produce catch the position cover the gold plaque material.The example of suitable polymkeric substance comprises for example polyolefine and fluorinated polymer.Usually before using, the solid phase material washing is desalted and other impurity to remove.It can stored dry or is stored in the aqeous suspension standby.Preferred solid phase material is used for the container that circulates, such as in for example transfer pipet, syringe or stake, microtiter plate or the microfluidic device, though can also use the suspension method that does not relate to these containers.

The solid phase material that can be used for method of the present invention can comprise the multiple material of various ways.For example, it can be particle loose or that be fixed or globule form, fiber, foam, frit, microporous membrane, film or has the substrate form on little duplicating (microreplicated) surface.If solid phase material comprises particle, preferably they be uniformly, globular and inflexible, to guarantee good fluid flow characteristics.

Use for circulation of the present invention, this material is generally loose porous network form, is used to allow macromole to pass in and out equably and with being without prejudice, and is used to the surface-area that provides bigger.Preferably, for this application, solid phase material has than higher surface area, such as for example, surpasses 1 meters squared per gram (m ²/ g).For not relating to the application of using fluid means, solid phase material can the yes or no porous matrix.Therefore, film also can be used in some method of the present invention.

For the application of using particle or globule, they can be incorporated in the sample or sample be incorporated in the bed of particle/globule and and therefrom remove by for example centrifugal.Perhaps, can pass through several different methods (as, spraying drying) with particle/globule coating (as, figure coating) to the inert substrate that randomly scribbles tackiness agent (as, polycarbonate or polyethylene) on.If expectation can be duplicated substrate is little, be used to increase surface-area and strengthen and purify.Can also carry out pre-treatment with oxygen plasma, electron beam or uv-radiation, heating or corona treatment method.Substrate can be used as for example mulch film on the bank of microfluidic device, or is laminated on the mulch film.

In one embodiment, solid phase material comprises fibril-matrix, and it can have or not be absorbed in wherein particle.Fibril-matrix can comprise any in the multiple fiber.Usually, fiber is insoluble at aqueous environment.Its example comprises glass fibre, polyolein fiber (particularly polypropylene and polyethylene primitive fiber, Kevlar, fluorinated polymers fibres (particularly polytetrafluoroethylene fiber) and natural cellulosic fibre.Can use the mixture of fiber, its combination for nucleic acid can be active or inactive.Preferably, fibril-matrix formation has at least about 15 microns and is about 1 millimeter to the maximum, more preferably is the tablet of about 500 micron thickness to the maximum.

If the use particle, then particle is normally insoluble in aqueous environments.They can be made by the combination of a kind of material or multiple material, as in the coating particle.They can be expandable or nondistensible, though preferably they are nondistensible in water and organic liquid.Preferably, if particle is used to adhere to, it is made by non-bloating hydrophobic material.They can be selected according to the affinity to nucleic acid.The example of the expandable particle of some water is at United States Patent (USP) 4,565, describes among 663 people such as () Errede, 4,460,642 people such as () Errede and 4,373,519 people such as () Errede.Nondistensible particle is at United States Patent (USP) 4,810 in water, describes among 381 people such as () Hagen, 4,906,378 people such as () Hagen, 4,971,736 people such as () Hagen and 5,279,742 people such as () Markell.Preferred particle is a polyolefin particles, as polypropylene particles (as, powder).Can use the mixture of particle, its combination for nucleic acid can be active or inactive.

If use the coating particle, coating is preferably water insoluble and be insoluble to the nondistensible material of organic solvent.Coating can be adhered to or non-cohesive nucleic acid.Therefore, the fundamental particle of coating can be inorganic or organic.Fundamental particle can comprise the inorganic oxide that its covalent attachment is had organic group, as silicon-dioxide, aluminum oxide, titanium dioxide, zirconium white etc.For example, can use covalently bound organic group, as have the aliphatic group of different chain length (C2, C4, C8 or C18 group).

The example of suitable solid phase material that comprises fibril-matrix is at United States Patent (USP) 5,279,742 (people such as Markell), 4,906,378 (people such as Hagen), 4,153,661 (people such as Ree), 5,071,610 (people such as Hagen), 5,147,539 (people such as Hagen), 5,207,915 (people such as Hagen) and 5, describe among 238,621 people such as () Hagen.This material can be available from 3M Company (St.Paul, MN), trade name is SDB-RPS (Styrene-Divinyl Benzene Reverse PhaseSulfonate, 3M Part No.2241), positively charged ion-SR film (3M Part No.2251), C-8 film (3M Part No.2214) and negatively charged ion-SR film (3M Part No.2252).

Preferably include those of tetrafluoroethylene matrix (PTFE) especially.For example, United States Patent (USP) 4,810,381 (people such as Hagen) disclose solid phase material, it comprises: the tetrafluoroethylene fibril-matrix, with the nondistensible adsorptivity particle that is trapped in the matrix, the weight ratio of wherein nondistensible adsorptivity particle and tetrafluoroethylene is about 19: 1 to 4: 1, and other wherein compound solid phase material has the clean surface energy of 20 to 300 milli Newton/meter.United States Patent (USP) RE36,811 (people such as Markell) disclose the solid phase extractions medium, it comprises: fibril-matrix, with the adsorptivity particle that is trapped in the matrix, wherein particle comprises the porousness organic filler above 30 to maximum 100 weight %, with the porousness that is less than 70 to 0 weight % (applying organic layer or uncoated) inorganic particulate, the weight ratio of adsorptivity particle and PTFE is 40: 1 to 1: 4.

Particularly preferred solid phase material derives from 3M Company, and St.Paul is under the trade name EMPORE of MN.The ultimate principle of EMPORE technology is to use any adsorber particles to produce the ability that load has the film or the dish shape thing of particle.(90% sorbent material: 10%PTFE's particle closely combines in by weight) at the inert base of tetrafluoroethylene.The PTFE protofibril hinders the activity of particle never in any form.The extraction medium that EMPORE film manufacturing process produces is compared with the medium by the particle preparation of identical size that uses in conventional solid phase extractions (SPE) post or cylinder, can obtain finer and close, more uniform medium.

In a further preferred embodiment, solid phase (as, microporosity thermoplastic polymer carrier) have with connect by protofibril isolated, random dispersion, have inhomogeneous shape, each to etc. big a plurality of thermoplastic polymer particles microporosity structure that is feature.The particle each interval provides the network of micropore between them.Particle is connected to each other by the protofibril from each bombardment to adjacent particles.In particle or the protofibril one or both can be hydrophobic.Preferred this examples of material has higher surface area, as often restraining and the most about 5 micron pore size up to 40 meter 2/ by mercury surface-area commercial measurement.

Such filamentary material can utilize the isolating optimization technique of induction phase to produce by relating to.This relates under the temperature that enough forms uniform mixture with immiscible liquid melt-mixing thermoplastic polymer, form goods with desired shape from the solution form, formed article is cooled off so that induce being separated of liquid and polymkeric substance, and polymer cure and the liquid of removing signal portion stay the microporosity polymeric matrix the most at last.This method and preferable material be at United States Patent (USP) 4,726, describes in detail among 989 (Mrozinski), 4,957,943 people such as () McAllister and 4,539,256 (Shipman).This material is called thermic phase separation membrane (TIPS film) and is particularly preferred.

Other suitable solid phase material is included in United States Patent (USP) 5,328, disclosed nonwoven material among 758 (people such as Markell).This material comprises and contains compression or merge the non-woven tablet of particulate (primitive fiber of preferred blowing) that it comprises the chromatographic grade particle of high adsorption efficiency.

Other suitable solid phase material comprises those that are called as HIPE Foams, and it is described in for example U.S. Patent Publication 2003/0011092 people such as () Tan." HIPE " or " High Internal Phase Emulsion " be meant comprise the active phase (being generally oil phase) of successive and not with the miscible discontinuous phase of oil phase or the emulsion of common external phase (being generally water), wherein immiscible phase accounts at least 74 volume % of emulsion.Many foam of polymers of being made by HIPE are generally relative perforate.This means that great majority or all adjacent holes of Kong Douyu communicate unblockedly.Have window between the Kong Zaikong of the foamy structure of this perforate basically, it is enough big usually, transfers to another hole from a hole to allow liquid in foamy structure.

Solid phase material can comprise the position of catching that is used to catch inhibitor.In this article, " catching the position " is meant that the group that is covalently attached on the solid phase material (as, functional group) or non-covalent (as, hydrophobicity) are incorporated into the molecule on the solid phase material.

Preferably, solid phase material comprises the functional group that catches inhibitor.For example, solid phase material can comprise chelation group.Here, " chelation group " for multiple-limb and can with atoms metal or ion (though inhibitor may by or may not remain on the solid phase material by chelating mechanism) form those of chelate complexes.The introducing of chelation group can realize by multiple technologies.For example, nonwoven material can hold with the functionalized globule of chelation group.Perhaps, the fiber of nonwoven material can directly carry out functionalized with chelation group.

The example of chelation group for example comprises-(CH ₂-C (O) OH) ₂, three (2-amino-ethyl) amine groups, iminodiacetic acid (salt) acid groups, nitrilotriacetic acid(NTA) group.Can chelation group be incorporated in the solid phase material by multiple technologies.They can be introduced into by the chemosynthesis of material.Perhaps, can with polymer-coated (as the coating of, figure) that comprise required chelation group to inert substrate (as, polycarbonate or polyethylene) on.If expectation can be duplicated substrate is little, be used to increase surface-area and strengthen and purify.Can also use oxygen plasma, electron beam or uv-radiation, heating or the pre-treatment of corona treatment method.Substrate can be used as for example mulch film on the bank of microfluidic device, or is laminated to mulch film.

The chelating solid phase material is commercially available and can be used as solid phase material in the present invention.For example, for certain embodiments of the present invention, preferably include the EMPORE film of chelation group such as iminodiethanoic acid (for the form of sodium salt).The particle of this film is at United States Patent (USP) 5,147, and is open and can be used as EMPORE Extraction Disks (47mm, No.2271 or 90mm are No.2371) available from 3M Company among 539 people such as () Hagen.For certain embodiments of the present invention, preferably include the EMPORE film of the ammonium derivatize of chelation group.For making dish shape thing be in ammonium form, its 0.1M ammonium acetate buffer with 50mL (pH 5.3) can be washed, wash with several reagent waters subsequently.

Other chelating examples of material includes but not limited to crosslinked polystyrene beads, and it can derive from Bio-Rad Laboratories, and (Hercules is under trade name CHELEX CA) for Inc.; Crosslinked agarose beads with three (2-amino-ethyl) amine, iminodiethanoic acid, nitrilotriacetic acid(NTA), polyamine and poly-imines; And available from Rohm and Haas (Philadelphia, trade name DUOLITE C-467 PA) and the chelating ion exchange resin under the DUOLITE GT73; AMBERLITE IRC-748, DIAION CR11, DUOLITE C647.

CHELEX 100 resins are for comprising iminodiethanoic acid ester group (N-(CH ₂-C (O) OH) ₂) the SDVB copolymer, it has high affinity for polyvalent metal ion and can be used in some method of the present invention, though do not cater to the need so in the method for implementing in microfluidic device.

Usually, the desired concn density of the chelation group on the solid phase material be about 0.02 receive rub/square millimeter, though think that wide range of concentrations density is possible.

The capture material of other type comprises anion-exchange material, cation exchange material, activated carbon, anti-phase, positive, vinylbenzene-Vinylstyrene, aluminum oxide, silicon-dioxide, zirconium white and metallic colloid.The example of suitable anion-exchange material comprises strong anion exchanger such as quaternary ammonium, dimethylethanolamine, quaternary alkylamine, tri methyl benzyl ammonium and the dimethyl ethanol hexadecyldimethyl benzyl ammonium that is generally chloride form; With weak anionite, as polyamines.The example of suitable cation exchange material comprises strong cationite such as sulfonic acid, is generally sodium-salt form; With weak cationite such as carboxylic acid, be generally the form of acid.The example of suitable carbonaceous material comprises EMPORE carbon material, carbon pearl.Suitable anti-phase C8 and C18 examples of material comprise with octadecyl or octyl group carries out end capped silica bead and has C8 and the EMPORE material of C18 silica bead (deriving from 3M Co., St.Paul, the EMPORE material of MN).The positive examples of material comprises hydroxyl and dihydroxyl.

Also commercially available material can be carried out modification or be directly used in the method for the present invention, in microfluidic device.For example, for example, can trade name LYSE ANDGO (Pierce will be derived from, Rockford, IL), RELEASE-IT (CPG, NJ), GENE FIZZ (Eurobio, France), GENE RELEASER (Bioventures Inc., Murfreesboro, TN) and BUGS N BEADS (GenPoint, Oslo, solid phase material Norway), and Zymo globule (Zymo Research, Orange is CA) with Dynal globule (Dynal, Oslo, Norway) be attached in the method for the present invention as the solid-phase capture material, particularly be attached in the microfluidic device.

In some embodiment of this method, solid phase material comprises coating reagent.Preferred coatings reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration.In some embodiment of this method, solid phase material comprises the tetrafluoroethylene fibril-matrix, be absorbed in the adsorptivity particle in the matrix and be coated on coating reagent on the solid phase material, and wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration.

The example of suitable tensio-active agent is listed below.

Suitable alkaline example comprises NaOH, KOH, LiOH, NH ₄OH, and primary amine, secondary amine or tertiary amine.

The example of suitable polyelectrolyte comprise polystyrolsulfon acid (as, poly-(4-Sodium styrene sulfonate) or PSSA), polyvinyl phosphonic acids, polyvinyl boric acid, polyvinylsulfonic acid, polyvinyl sulfuric acid, polystyrene phosphonic acids, polyacrylic acid, polymethyl acrylic acid, sulfonated lignin, carrageenin, heparin, chondroitin sulfate (chondritin sulfate) and salt or other derivative.

The example of the polymkeric substance barrier of suitable alternative infiltration comprises polymkeric substance such as acrylate, acrylamide, azlactone, polyvinyl alcohol, polymine, polysaccharide.This polymkeric substance can be various ways.They can be water miscible, water is expandable, water-insoluble, form of hydrogels etc.For example, the polymkeric substance barrier can be prepared as and make it play as the effect than macroparticle such as white corpuscle, nucleus, virus, bacterium and nucleic acid such as human gene group DNA and proteinic strainer.These surfaces can be by those skilled in the art by suitably selecting functional group, being suitable for separating according to size and/or electric charge by crosslinked etc. the transformation.This material is buied easily or can easily be prepared by those skilled in the art.

Preferably, solid phase material is inexcessively washed away any tensio-active agent with tensio-active agent coating, though can wash other coating reagent off if desired.Usually, coating can several different methods be carried out, as dipping, roller coat, spraying etc.Usually before using, will be loaded with the solid phase material of coating reagent then at for example air drying.

What need especially is to scribble tensio-active agent, preferably scribble the solid phase material of nonionic surface active agent.This can realize according to the operation of mentioning in the embodiment part.Though do not plan to be limited by theory, think that the adding of tensio-active agent increases the wettability of solid phase material, its permission inhibitor penetrates in the solid phase material and with it and combines.

The coating reagent that is preferred for solid phase material is a group water solution, though with an organic solvent (alcohol etc.) if desired.The coating reagent heap(ed) capacity is should be enough high, makes the sample solid phase material that can drench.Yet it can not be too high, makes coating reagent itself by remarkable wash-out.Preferably, if coating reagent nucleic acid wash-out contains the coating reagent that is no more than about 2 weight % in the sample of wash-out.Usually, the concentration of coating solution in solution, can hang down the coating reagent that reaches 0.1 weight % and in solution up to the coating reagent of 10 weight %.

Tensio-active agent

Nonionic surface active agent.It is known that multiple suitable nonionic surface active agent is arranged, and it can be used as lytic reagent (above-mentioned discussion), eluent (following discussion) and/or as the coating on the solid phase material.They comprise for example polyoxyethylene surfactant, carboxylicesters tensio-active agent, carboxylic acid amide tensio-active agent etc.Commercially available nonionic surface active agent comprises the n-dodecane acyl sucrose; dodecyl-β-D-glycopyranoside; n-octyl-β-D-pyrans maltoside; n-octyl-β-D-sulfo-glycopyranoside; the n-dodecane acyl sucrose; positive decyl-β-D-pyrans maltoside; positive decyl-β-D-sulfo-maltoside; n-heptyl-β-D-glycopyranoside; n-heptyl-β-D-sulfo-glycopyranoside; n-hexyl-β-D-glycopyranoside; n-nonyl-β-D-glycopyranoside; positive capryloyl sucrose; n-octyl-β-D-glycopyranoside; cyclohexyl-n-hexyl-β-D-maltoside; cyclohexyl-N-methyl-β-D-maltoside; digitonin; with derive from trade name PLURONIC; TRITON; under the TWEEN those, and many other commercially available and that in Kirk Othmer Technical Encyclopedia, enumerate those.Its example is enumerated in following table 1.Preferred surfactants is a polyoxyethylene surfactant.Preferred tensio-active agent comprises octylphenoxy polyethoxy ethanol.

Table 1

The tensio-active agent trade(brand)name	Nonionic surface active agent	Supplier
			PLURONIC F127	The alcohol and/or the propenoxylated straight chain alcohol of the ethoxylation of modification	Sigma St.Louis，MO
TWEEN 20	Polyoxyethylene (20) mono laurate sorbitan ester	Sigma St.Louis，MO
			TRITON X-100	Uncle's octylphenoxy polyethoxy ethanol	Sigma St.Louis，MO
BRIJ 97	Polyoxyethylene (10) oleyl ether	Sigma St.Louis，MO
			IGEPAL CA-630	Octylphenoxy gathers (vinyloxy group) ethanol	Sigma St.Louis，MO
TOMADOL 1-7	Ethoxylated alcohol	Tomah Products Milton，WI
			Vitamin E TPGS	D-alpha-tocopherol cetomacrogol 1000	Eastman Kingsport，TN

Also suitable is the fluorinated nonionic type tensio-active agent of disclosed type in U.S. Patent Publication 2003/0139550 (people such as Savu) and 2003/0139549 (people such as Savu).Other non-ionic type fluorinated surfactant comprises (Wilmington, those under trade name ZONYL DE) available from DuPont.

Zwitterionics.Known multiple suitable zwitterionics, it can be used as coating on the solid phase material, as lytic reagent and/or as eluent.They comprise for example alkyl amido betaine and amine oxide, alkyl betaine and amine oxide thereof, sultaine, hydroxyl sulfo betaine, both sexes glycinate, both sexes propionic ester, equilibrated both sexes many carboxyls glycinate and alkyl polyamino glycinate.Protein has charged or uncharged ability according to pH, and therefore at suitable pH, preferred pI as the bovine serum albumin or the chymotrypsinogen of modification, can be used as zwitterionics for the protein of about 8-9.The object lesson of zwitterionics is the courage amidopropyl dimethyl propylene ammonium sulphonate that derives under the trade name CHAPS of Sigma.Preferred tensio-active agent comprises N-dodecyl-N, N-dimethyl-3-ammonium-1-propane sulfonate.

Cats product.Known multiple suitable cats product, it can be used as lytic reagent, eluent and/or as the coating on the solid phase material.They comprise for example quaternary ammonium salt, polyoxyethylene alkylamine and alkyl amine oxide.Usually, suitable quaternary ammonium salt comprises the group of at least one higher molecular weight and the group of two or three lower molecular weights, they are connected in shared nitrogen-atoms with the generation positively charged ion, and wherein the negatively charged ion of electronic equilibrium is selected from halogenide (bromide, muriate etc.), acetate moiety, nitrite anions and low alkyl group sulfonate radical (methanesulfonate etc.).Higher molecular weight substituting group on the nitrogen is often for comprising about 10 senior alkyls to about 20 carbon atoms, the substituting group of lower molecular weight can be has 1 to the alkyl of about 4 carbon atoms such as the low alkyl group of methyl or ethyl, it can be substituted in some cases, as being replaced by hydroxyl.One or more substituting groups can comprise aryl moiety or can be replaced by aryl, as benzyl or phenyl.Possible lower molecular weight substituting group also can comprise about 1 low alkyl group to about 4 carbon atoms, as methyl and ethyl; The substituted rudimentary poly-alkoxyl group part that has a hydroxyl end groups as the polyoxyethylene part, and has following general formula:

R (CH ₂CH ₂O) _(n-1)CH ₂CH ₂OH wherein R is (C1-C4) divalent alkyl that is incorporated on the nitrogen, and n represents about 1 to about 15 integer.Perhaps one or two this rudimentary poly-alkoxyl group with terminal hydroxyl can directly be incorporated into quaternary nitrogen, rather than is incorporated into nitrogen by aforementioned low alkyl group.The example that is used for useful quaternary ammonium halides tensio-active agent of the present invention includes but not limited to derive from methyl-two (2-hydroxyethyl) cocoyl ammonium chlorides or oleyl ammonium chloride (being respectively ETHOQUAD C/12 and O/12) and methyl polyoxyethylene (15) octadecyl ammonium chloride (ETHOQUAD 18/25) of Akzo Chemical Inc..

Anion surfactant.Known multiple suitable anion surfactant, it can be used as lytic reagent, eluent and/or as the coating on the solid phase material.Spendable aniorfic surfactant comprises sulfonate and vitriol, as alkyl-sulphate, sulfated alkyl ether, alkylsulfonate, alkylether sulfonate, alkylbenzene sulfonate, alkylbenzene ether sulfate, alkyl sulfoacetate, secondary alkyl sulfonate, secondary alkyl sulfate etc.Have in these many can comprise the poly-alkoxylation group (as, ethylene oxide group and/or propylene oxide group, it can be the layout of random, orderly or block) and/or cationic counterion such as Na, K, Li, ammonium, protonated tertiary amine such as trolamine or quaternary ammonium group.Its example comprises: alkylether sulfonate, as available from Stepan Company, Northfield, the trade name POLYSTEP B12 of IL and the sodium lauryl ether sulphates under the B22, with available from Nikko Chemicals Co., Tokyo, the N-methyltaurine sodium under the trade name NIKKOL CMT30 of Japan; Available from Clariant Corp., Charlotte, the secondary alkyl sulfonate under the trade name HOSTAPUR SAS of NC, it is (C14-C17) Seconary Alkane Sulphonate Sodium (sulfonated); Methyl-2-sulfo group alkyl ester is as methyl-2-sulfo group (C12-C16) ester sodium with available from 2-sulfo group (C12-C16) the lipid acid disodium under the trade name ALPHASTE PC-48 of Stepan Company; As all available from the dodecyl sulfoacetic acid sodium (trade name LANTHANOL LAL) of Stepan Co. and alkyl sulfoacetate and the alkyl sulfo succinate under the MAKABATE LO 100 (trade name STEPANMILD SL3); And alkyl-sulphate, as available from the ammonium lauryl sulfate under the trade name STEPANOL AM of Stepan Co..

Another kind of useful anion surfactant comprises phosphoric acid salt, as alkylphosphonic, alkyl ether phosphate, aralkyl phosphoric acid salt and aralkyl ethers phosphoric acid salt.Have in these many can comprise poly-alkoxy base (as, ethylene oxide group and/or propylene oxide group, its can be random, in order or the layout of block).That its example comprises is single-, two-and three-(alkyl glycerylether)-o-phosphoric acid ester, typically refer to available from the trilaureth-4-phosphoric acid salt under the trade name HOSTAPHAT 340KL of Clariant Corp.; With available from Croda Inc., Parsipanny, PPG-5 ceteth 10 phosphoric acid salt under the trade name CRODAPHOS SG of NJ; And alkyl and alkylamidoalkyl alkyl dialkylamine oxide compound.The example of amine oxide tensio-active agent comprises those (all available from the Stepan Co.) under trade name AMMONYX LO, LMDO and the CO, and it is laurylamide base propyl-dimethyl amine oxide and hexadecyl amine oxide.

Elution technique

Be used to keep the embodiment of the solid phase material of inhibitor for use, the more spissated zone of the sample of material that can use multiple eluent wash-out to comprise to contain nucleic acid (as, nucleus) and/or the nucleic acid that discharges.This eluent can comprise water (preferred not the water of qiagen rnase enzyme), damping fluid, tensio-active agent (it can be cationic, anionic, non-ionic type or amphoteric ionic surfactant) or highly basic.

Preferably, eluent is (that is, pH is greater than 7) of alkalescence.For some embodiment, the pH of eluent is at least 8.For some embodiment, the pH of eluent is for the highest by 10.For some embodiment, the pH of eluent is for the highest by 13.If the nucleic acid of wash-out is directly used in amplification procedure such as PCR, then eluent should be formulated as and makes that each component concentrations can inhibitory enzyme (as, Taq polysaccharase) or stop amplified reaction.

The example of suitable tensio-active agent comprises above-mentioned those, particularly, and for being called as those of SDS, TRITON X-100, TWEEN, fluorinated surfactant and PLURONICS.Tensio-active agent is provided in group water solution usually, though with an organic solvent (alcohol etc.) if desired.In the preferred eluent surfactant concentrations minimum based on the gross weight of eluent be 0.1 weight/volume % (w/v%).In the preferred eluent surfactant concentrations based on the gross weight of eluent for being not more than 1w/v%.Can randomly stablizer such as polyoxyethylene glycol be used with tensio-active agent.

The example of suitable elution buffer comprises TRIS-HCl, N-[2-hydroxyethyl] piperazine-N '-[2-ethanesulfonic acid] (HEPES), the 3-[N-morpholino] propanesulfonic acid (MOPS), piperazine-N, N '-two [2-ethanesulfonic acid] (PIPES), the 2-[N-morpholino] ethyl sulfonic acid (MES), TRIS-EDTA (TE) damping fluid, Trisodium Citrate, ammonium acetate, carbonate and supercarbonate etc.

Preferably, in the eluent concentration of elution buffer minimum be 10 millis rub (mM).Preferably, surfactant concentrations is not more than 2 weight % in the eluent.

Usually, preferably use basic solution to realize containing the wash-out of the nucleic acid of the material of nucleic acid and/or release.Though do not plan to be bound by theory, think that and wash out-of-phase with water relatively, basic solution can improve the combination of inhibitor.Basic solution also promotes to contain the cracking of the material of nucleic acid.Preferably, the pH of basic solution is 8 to 13, more preferably 13.The example in the source of high pH comprises the aqueous solution of NaOH, KOH, LiOH, quaternary nitrogen based hybroxide, tertiary amine, secondary amine or primary amine etc.If basic solution is used for wash-out, usually it is neutralized with for example TRIS damping fluid in step subsequently, be ready for the sample of PCR with formation.

The use of basic solution can optionally destroy RNA, with permission DNA is analyzed.Otherwise, can in preparation, add rnase so that the RNA deactivation, subsequently with the hot deactivation of rnase.Similarly, can add deoxyribonuclease is used for optionally destroying DNA and allows RNA is analyzed; Yet, can with do not destroy RNA other lysis buffer (as, TE) be used for this method.Can also add ribonuclease inhibitor such as RNAsin to the preparation of the RNA preparation that is used for experiencing PCR in real time.

Wash-out at room temperature carries out usually, though higher temperature can produce higher yield.For example, if desired, the temperature of eluent can be up to 95 ℃.Wash-out carried out in 10 minutes usually, though preferred 1-3 minute elution time.

Device and test kit

The example that is used for the device of method of the present invention comprises by company such as Millipore, Inc. (Bedford, MA), Bio-Rad, Inc. (Hercules, CA), Osmonics, Inc. (Westborough, MA) and Whatman Inc. (Clifton, the standard laboratory filter mounting that NJ) provides.Method of the present invention can promote in the mode by centrifugal, suction, pressure to carry out in the filtration unit (being called circulation device) of solution by the solid phase material motion.Other device comprises microtiter plate and microfluidic device.

The present invention also provides test kit, the teachings that it could include or not have the solid phase material, lytic reagent (particularly tensio-active agent such as nonionic surface active agent, pure or in solution) of support (for example filter mounting such as syringe filter support or centrifugal filter support or be used for having in each end the post of the frit that keeps the used material of particle) and be used for binding inhibitors and wash-out nucleic acid.Preferably, the invention provides and comprise the circulation container (more preferably, microfluidic device) test kit has solid phase (preferred polymers) material, and preferably has nonionic surface active agent in the circulation container.

Other component that can comprise in the test kit of the present invention comprises conventional reagent, as washing lotion, coupling buffer, cancellation damping fluid, sealing damping fluid, elution buffer, or the like.Other assembly that can comprise in the test kit of the present invention comprises conventional equipment, as centrifugal post, tube core, 96 hole filter plates, needle-based strainer, collector unit, syringe, or the like.

Test kit generally includes wrapping material, and it is meant one or more physical structures that are used to hold the test kit content.Wrapping material can be constructed by known method, are preferred for the environment that does not contain pollutent that provides aseptic.Wrapping material can have the label that is used for visualizingre agent box content.In addition, test kit comprises and is used to indicate teachings how to use material in the test kit.As used in this article, term " packing " is meant solid substrate or material, as glass, plastics, paper, paper tinsel etc.

" teachings " generally includes clear and definite expression, different methods of the present invention is described, comprise cracking condition (as, lytic reagent type and concentration), reagent and want relative quantity, the reagent/specimen mixture of blended sample hold-time, temperature, buffer condition, or the like.

Though aforesaid method can be used in the various devices, the various exemplary of preferred device are described in U.S. Patent Publication 2002/0047003 (2003 4 about 25 days open, people such as Bedingham).

The preferred embodiment that is used for method of the present invention comprises microfluidic device.Their common uses are furnished with the agent structure of incorporate microfluidic channel network therein.Aspect preferred, the agent structure of microfluidic device comprises the aggregate of two or more separate layers, described two or more separate layer forms microfluidic device of the present invention when suitably cooperating or combining, as comprises described passage and/or chamber herein.Usually, useful microfluidic device comprises top portion, bottom branch, internal portion, and wherein internal portion defines the passage and the chamber of device basically.Usually, the chamber comprises valve (as, valve partition) and is called the chamber that valve is arranged.

The particularly preferred device that is used for some embodiment of this paper is called control valve device, open in the applicant's that its title of submitting on December 12nd, 2003 is " Variable Valve Apparatus and Method " transferee's the U.S. Patent application co-pending 10/734,717.In this control valve device, valve arrangement allows to remove the selected part that is loaded into the specimen material in the treatment chamber (that is the treatment chamber that variable valve, is arranged).Removing by form opening in the valve partition in the predetermined position of selected part realizes.

The preferred valve partition is enough big, to allow the position according to the adjustment of features opening of specimen material in the treatment chamber.If rotary sample treatment unit after forming opening, then the material of the selected part of more close turning axle leaves treatment chamber by opening.The rest part of specimen material can not leave by opening, because its ratio open is farther apart from turning axle.

Can be in the valve partition in certain position according to one or more features of the specimen material that in treatment chamber, detects and form opening.The preferably treatment chamber comprises the detection window of the treatment chamber that light is imported into and/or spread out of.The feature of the specimen material that detects can comprise for example free surface of specimen material (capacity of specimen material in the characterization process chamber).Be in the valve partition at the radially outer selected distance of free surface and form opening, the ability of removing the specimen material of selected capacity from treatment chamber can be provided.

For specimen material that can be separated into different components such as whole blood, the rotation of sample processing device can cause separating of blood plasma and red blood cell component, thereby optionally removes component in for example different treatment chambers.

In some embodiments, might form opening by the select location place in one or more valve partitions and remove selected specimen material aliquot sample.Selected aliquot sample capacity can be measured according to the cross-sectional area of the treatment chamber between radial distance between the opening (measuring with respect to turning axle) and the opening.

Opening in the preferred valve partition forms under the situation that does not have the physics contact, as, by laser ablation, focused light heating etc.As a result, be preferably formed opening and do not penetrate the outermost layer of sample processing device, thereby limited the possibility of specimen material from the sample processing device seepage.

In one aspect, the present invention uses in sample processing device (as microfluidic device) the valve treatment chamber, have valve handle (as, heating, mixing, cracking, mixed stream) chamber comprises the treatment chamber with chamber enclosure, chamber enclosure is between the first and second relative major opposing sides of sample processing device, wherein treatment chamber occupies the process chamber area in the sample processing device, and wherein process chamber area have length and with the vertical width of length, and in addition wherein length greater than width.The treatment chamber that variable valve is housed also comprises the valve chamber that is positioned at process chamber area, valve chamber is between second major opposing side of chamber enclosure and sample processing device, wherein make valve chamber and treatment chamber isolation by the valve partition that valve chamber and treatment chamber are separated, and wherein the part of chamber enclosure between first major opposing side of valve partition and sample processing device.Detection window is positioned at process chamber area, and wherein detection window can see through selected electromagnetic energy, and this selected electromagnetic energy is introduced into and draws chamber enclosure.

In yet another aspect, the invention provides the method that permission is removed a part of sample from the treatment chamber selectivity that variable valve is housed.This method comprises provides aforesaid sample processing device (as, microfluidic device), sampling material in treatment chamber; Detect the feature of specimen material in the treatment chamber by detection window; Be in the valve partition at the select location along treatment chamber length and form opening, wherein Xuan Ding position is relevant with the specimen material feature of detection.This method also comprises by the opening that forms in the valve partition only to be transferred to a part of specimen material the valve chamber from treatment chamber.

Illustrative methods 1

In one embodiment, the invention provides from the method for sample separation nucleic acid.In this illustrative methods, inhibitor packages is contained in the cell, but should be appreciated that, situation not always not like this, method of the present invention can be used this sample.

This method comprises: provide to comprise material that contains nucleic acid and the sample of the cell that contains inhibitor (the described material that contains nucleic acid can be identical or different with the cell that contains inhibitor); Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor; Form the lysate sample enrichment region; Wherein the lysate sample enrichment region comprises material and the inhibitor that contains nucleic acid; The lysate sample enrichment region is separated basically with inferior enrichment region; Make isolating lysate sample enrichment region contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; Before making isolating sample concentration district contact solid phase material, simultaneously or the material cracking that randomly further will contain nucleic acid afterwards to discharge nucleic acid; Separate with the solid phase material that has adhered at least a portion inhibitor with the material and/or the nucleic acid that make at least a portion contain nucleic acid.

The material that contains nucleic acid can be identical or different with the cell that contains inhibitor, though they are normally different.That is to say that the material that contains nucleic acid may be identical with the cell that contains inhibitor.For example, if sample is a buffy coat, the material that then contains nucleic acid can be white corpuscle, and it comprises nucleus and inhibitor simultaneously.Wherein the nuclear lytic reagent of not cracking if use the leukocytic cytolemma of cracking (as, nonionic surface active agent), release inhibitor and nucleus is intact then, it also is considered the material that contains nucleic acid defined herein.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: the sample that comprises the cell that contains inhibitor and contain nuclear cell (described contain the cell of inhibitor and contain nuclear cell can be identical or different) is provided; Make biological sample effectively destroy cytolemma and discharge nucleus and inhibitor and form under the condition of lysate sample and contact nonionic surface active agent; Form the lysate sample enrichment region; Wherein the sample concentration district comprises nucleus and inhibitor; The lysate sample enrichment region is separated basically with sample time enrichment region; Make isolating lysate sample enrichment region contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; Before making isolating sample concentration district contact solid phase material, simultaneously or afterwards randomly further lysing cell nuclear to discharge nucleic acid; Separate with the solid phase material that has adhered at least a portion inhibitor with the material and/or the nucleic acid that make at least a portion contain nucleic acid.

Containing the cell of inhibitor and contain nuclear cell can be identical or different, though they are generally different cells.That is to say that containing nuclear cell may be identical with the cell that contains inhibitor.For example, inhibitor can comprise nucleoprotein and cell protein.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: provide microfluidic device, the separate chamber that it comprises feed compartment, the valve treatment chamber is arranged and comprise solid phase material; Provide and comprise material that contains nucleic acid and the sample of the cell that contains inhibitor (the described material that contains nucleic acid can be identical or different with the cell that contains inhibitor); Sample is placed feed compartment; Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor; Lysate sample has been transferred to the valve treatment chamber; Form the lysate sample enrichment region in the valve treatment chamber is arranged, wherein the lysate sample enrichment region comprises material and the inhibitor that contains nucleic acid; The lysate sample enrichment region is separated basically with inferior enrichment region; Isolating lysate sample enrichment region is transferred to the separate chamber contact, make at least a portion inhibitor preferentially be attached to solid phase material with solid phase material; Wherein solid phase material comprises and catches position (preferred chelating functional group), is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; Before isolating sample concentration district contact solid phase material, simultaneously or afterwards randomly further cracking contain the material of nucleic acid to discharge nucleic acid; Separate with the solid phase material that has adhered at least a portion inhibitor with the material and/or the nucleic acid that make at least a portion contain nucleic acid.As discussed above, the material that contains nucleic acid can be identical or different with the cell that contains inhibitor, though they are normally different.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: provide microfluidic device, the separate chamber that it comprises feed compartment, the valve treatment chamber is arranged and comprise solid phase material; The sample that comprises the cell that contains inhibitor and contain nuclear cell (described contain the cell of inhibitor and contain nuclear cell can be identical or different) is provided; Sample is placed feed compartment; Make sample effectively destroy cytolemma and discharge nucleus and the condition of inhibitor under contact nonionic surface active agent with the formation lysate sample; Lysate sample has been transferred to the valve treatment chamber; Form the lysate sample enrichment region in the valve treatment chamber is arranged, wherein the lysate sample enrichment region comprises nucleus and inhibitor; The lysate sample enrichment region is separated basically with inferior enrichment region; Isolating lysate sample enrichment region is transferred to the separate chamber contact, make at least a portion inhibitor preferentially be attached to solid phase material with solid phase material; Wherein solid phase material comprises and catches position (preferred chelating functional group), is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; Before isolating sample concentration district contact solid phase material, simultaneously or afterwards randomly further cracking contain the material of nucleic acid to discharge nucleic acid; Separate with the solid phase material that has adhered at least a portion inhibitor with the material and/or the nucleic acid that make at least a portion contain nucleic acid.As discussed above, containing the cell of inhibitor and contain nuclear cell can be identical or different, though their normally different cells.

In some embodiment of this method, the material that contains nucleic acid comprises nucleus, and this method comprises from solid phase material separation at least a portion nucleus.That is to say undamaged nucleus contact solid phase material.

In some embodiment of this method, separate the material that at least a portion contains nucleic acid (as, nucleus) and/or nucleic acid from solid phase material and comprise that the material that will contain nucleic acid (as, nucleus) and/or nucleic acid transfers in the amplified reaction chamber (as, PCR sample chamber).

In some embodiment of this method, the coating reagent on the solid phase material comprises tensio-active agent.Preferably, tensio-active agent is non-ionic type or amphoteric ionic surfactant, more preferably, is nonionic surface active agent.

In some embodiment of this method, separate material that at least a portion contains nucleic acid (as, nucleus) and/or nucleic acid and comprise material (as, nucleus) and/or the nucleic acid that contains nucleic acid with eluent from solid phase material wash-out at least a portion.

In some embodiment of this method of the present invention, if expectation, this method can comprise that the further use second lytic reagent cracking contains the material of nucleic acid (as, nucleus).

Perhaps, in some embodiment of this method, eluent also is a lytic reagent.For while wash-out and cracking, preferred highly basic is as NaOH.For a wash-out and not cracking, preferably water or TE damping fluid.Separate the wash-out of sample for presplitting, can use any elution reagent (that is eluent).

With reference to figure 1, be applicable to that the preferred embodiment of the microfluidic device of these embodiments comprises feed compartment 10, the mixing section of choosing wantonly 12, valve treatment chamber 14 is arranged, comprise separate chamber 16, eluent chamber 18, the waste chamber 20 of solid phase material and the amplified reaction chamber of choosing wantonly 22.The title that comprises the applicant's that this microfluidic device that the valve treatment chamber arranged was submitted on December 12nd, 2003 transferee is open in the U.S. Patent application co-pending 10/734,717 of Variable Valve Apparatus and Method.These chambers are fluid communication each other, make sample can be loaded into feed compartment 10, and sample can be transferred to mixing section 12 then, if perhaps it does not exist, then sample has been directly transferred in the valve treatment chamber 14.If sample for example with lytic reagent pre-mixing (that is, pre-cracking), then do not need mixing section 12.Perhaps, mixing section 12 can comprise lytic reagent, for example (and being generally exsiccant) form for presetting.Can sample in being arranged, valve treatment chamber 14 be concentrated by several different methods, usually by centrifugal.There is the valve of valve treatment chamber 14 to be arranged so that usually sample concentration district (it comprises the required material that contains nucleic acid and/or nucleic acid and inhibitor) can separate with sample time enrichment region (it also can comprise inhibitor and a small amount of required material that contains nucleic acid and/or nucleic acid) basically.Sample time enrichment region is transferred to waste chamber 20 usually.The more enrichment region of sample is transferred to separate chamber 16 and is used to contact solid phase material, makes at least a portion inhibitor preferentially be attached to solid phase material.Then the eluent in the eluent chamber 18 is transferred to separate chamber 16, to remove at least a portion required material that contains nucleic acid and/or nucleic acid.In some cases, nucleus can be stored again be used for after use.In certain embodiments, this material can be directly transferred to amplified reaction chamber 22, is used for for example carrying out the PCR process.Amplified reaction chamber 22 can comprise randomly that (and being generally exsiccant) of presetting is used for amplified reaction (as, reactant PCR).

Illustrative methods 2

In another embodiment, the invention provides from the method for sample separation nucleic acid.In this illustrative methods, inhibitor packages is contained in the cell, but should be appreciated that, situation not always not like this, method of the present invention can be used this sample.

This method comprises: the sample that comprises material that contains nucleic acid and the cell that contains inhibitor (it can be identical or different) is provided; Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor; Make lysate sample contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; Before making isolating sample concentration district contact solid phase material, simultaneously or afterwards randomly further cracking contain the material of nucleic acid to discharge nucleic acid; With separate with the solid phase material that has adhered at least a portion inhibitor by material and/or the nucleic acid that makes at least a portion contain nucleic acid with the eluent wash-out, condition is that eluent is not a nonionic surface active agent.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: the sample that comprises material that contains nucleic acid and the cell that contains inhibitor (they can be identical or different) is provided; Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor; Make lysate sample contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; The material that makes at least a portion contain nucleic acid separates with the solid phase material that has adhered at least a portion inhibitor; With after separating the material that contains nucleic acid, further cracking contains the material of nucleic acid to discharge nucleic acid.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: provide microfluidic device, the separate chamber that it comprises feed compartment and comprises solid phase material; The sample that comprises material that contains nucleic acid and the cell that contains inhibitor (they can be identical or different) is provided; Sample is placed feed compartment; Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor; Lysate sample is transferred to the separate chamber with the contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material; Before making sample contact solid phase material, simultaneously or afterwards randomly further cracking contain the material of nucleic acid to discharge nucleic acid; Wherein solid phase material comprises and catches position (as, chelating functional group), is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; With separate with the solid phase material that has adhered at least a portion inhibitor by material and/or the nucleic acid that makes at least a portion contain nucleic acid with the eluent wash-out, condition is that eluent is not a nonionic surface active agent.

In other embodiments, the invention provides from the method for sample separation nucleic acid, this method comprises: provide microfluidic device, the separate chamber that it comprises feed compartment and comprises solid phase material; The sample that comprises material that contains nucleic acid and the cell that contains inhibitor (they can be identical or different) is provided; Sample is placed feed compartment; Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor; Lysate sample is transferred to the separate chamber makes at least a portion inhibitor preferentially be attached to solid phase material with the contact solid phase material; Wherein solid phase material comprises and catches position (as, chelating functional group), is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; Separate with the solid phase material that has adhered at least a portion inhibitor with the material that makes at least a portion contain nucleic acid; With after separating the material that contains nucleic acid, further cracking contains the material of nucleic acid to discharge nucleic acid.

Discuss for illustrative methods 1 as above, the material that contains nucleic acid of the embodiment of illustrative methods 2 can be identical or different with the cell that contains inhibitor, though they are normally different.That is to say that the material that contains nucleic acid may be identical with the cell that contains inhibitor.Equally, in illustrative methods 2, sample can comprise free nucleic acid and free inhibitor.

In some embodiment of this method, from solid phase material separate the material contain nucleic acid (as, nucleus) (if and/or from containing the separating substances of nucleic acid, then for nucleic acid) comprise the material that will contain nucleic acid (as, nucleus) (and/or nucleic acid) is transferred in the amplified reaction chamber (as, PCR sample chamber).

In some embodiment of this method, solid phase material comprises the tetrafluoroethylene fibril-matrix, be trapped in the adsorptivity particle in the matrix and be applied to coating reagent on the solid phase material, and wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration.

In some embodiment of this method, separate and to comprise the material (as, nucleus) (and/or nucleic acid) that contains nucleic acid with eluent from solid phase material wash-out at least a portion.

With reference to figure 2, be applicable to that the preferred embodiment of the microfluidic device of these embodiments comprises feed compartment 30, optional mixing section 32, the separate chamber 36 that comprises solid phase material, eluent chamber 38 and optional amplification chamber 42.These chambers are fluid communication each other, make the sample can be loaded in the feed compartment 30, and sample is transferred to mixing section 32 then, perhaps if there is no mixing section 32, and then sample is directly transferred in the separate chamber 36.If sample for example with lytic reagent pre-mixing (that is, pre-cracking), then no longer need mixing section 32.Perhaps, mixing section 32 can comprise lytic reagent, for example (and being generally exsiccant) form to preset.Lysate sample transferred to be used to contact solid phase material in the separate chamber 36, make at least a portion inhibitor preferentially be attached to solid phase material.Then the eluent in the eluent chamber 38 is transferred to separate chamber 36, to remove at least a portion required material that contains nucleic acid and/or nucleic acid.Can use the highly basic elution samples, be used for further cracking.In some cases, after using the highly basic cracking, sample can be heated several minutes down at high temperature (95 ℃), making has inhibiting protein denaturation to PCR, uses the neutralization of reagent such as TRIS damping fluid subsequently, is used for regulating before PCR the pH of sample.Perhaps, can with the sample transfer of wash-out to other being used to use other method (as, heating, Proteinase K) further in the cracked chamber.Perhaps, if expectation, these reagent can be (and being exsiccant) of presetting.In certain embodiments, this material can be directly transferred to and be used for for example carrying out the PCR process in the amplified reaction chamber 42.Amplified reaction chamber 42 can randomly comprise preset be used for amplified reaction (as, reactant PCR).

Other embodiments

In some embodiment that relates to this method of using microfluidic device, place feed compartment to occur in to make sample to contact before first lytic reagent in sample.Perhaps, sample being placed feed compartment occur in sample contacts after first lytic reagent.First lytic reagent can be for example water or nonionic surface active agent.

Other if desired cracking can be used other cracking condition to discharge nucleic acid from the material that contains nucleic acid (as, nucleus).For example, it is included in the material experience highly basic that contains nucleic acid is handled.Highly basic is generally NaOH, but can be other highly basic, as KOH, LiOH, NH ₄OH, and primary amine, secondary amine or tertiary amine.Usually, temperature is a room temperature.If use alkali, the sample that then comprises the nucleic acid of release need be adjusted its pH, if particularly nucleic acid will experience subsequently amplification procedure.Therefore, certain embodiments of the present invention comprise that the pH that adjusts sample is usually at least 7.5, usually to being not more than 9.

In some embodiment that relates to this method of using microfluidic device, first lytic reagent is a nonionic surface active agent.In some embodiment of this method, feed compartment comprises first lytic reagent (as, (and being generally exsiccant) nonionic surface active agent that presets) and sample takes place when sample is placed feed compartment and contacts first lytic reagent.

Perhaps, can be in the treatment chamber that wherein has first lytic reagent (being preferably nonionic surface active agent) subsequently with sample transfer.For example, sample contacts first lytic reagent (being preferably nonionic surface active agent) and takes place in mixing section, carries out under mixing fully to destroy cytolemma and to discharge nucleus and inhibitor, to form lysate sample.Should be appreciated that if use " first " lytic reagent, this method must not used " second " lytic reagent; On the contrary, term used herein " first lytic reagent " is distinguished with any other lytic reagent that uses mutually if be.

As mentioned above, in damping fluid (particularly TRIS damping fluid), add sucrose and can help nuclear separation.Damping fluid also can comprise magnesium salts and tensio-active agent such as TRITON X-100.This also can be leukocytic cracking good medium is provided.In addition, in some cases, when the needs collecting cell is examined, particularly in microfluidic device, use nucleus storage damping fluid to come in handy.Nucleus storage damping fluid can comprise sucrose, magnesium salts, EDTA, dithiothreitol (DTT), 4-(2-amino-ethyl) benzene sulfonyl fluorine (AEBSF) and/or glycerine and allow nuclear stable state storage in for example damping fluid (as, TRIS damping fluid).

In some embodiment that relates to this method of using microfluidic device, in the valve treatment chamber is arranged, form the sample concentration district and comprise the sample in the treatment chamber centrifugal.Usually, sample concentration district and sample time enrichment region is separated comprise sample time enrichment region is transferred to waste chamber by valve.When using microfluidic device, the centrifugal speed of for example keeping 2 minutes 400rcf is generally desirable, although can use higher rotating speed and longer rotational time.

Nonionic surface active agent such as TRITON X-100 can be preset (and if expectation can be equably dry) in the mixing section of microfluidic device, for example make and cracking to take place when sample (as, blood) during with the tensio-active agent mixed for several minutes.In other cases, before can be in being incorporated into mixing section with the dilute solution of tensio-active agent and sample (as, blood) pre-mixing.

In other cases, when not using nonionic surface active agent, can make water or ammonium chloride be used for erythrocytic cracking.In mixing section, take place after the cracking, in low relatively speed that white corpuscle is centrifugal down.

In some embodiment of described method, sample can be whole blood in this article.Then usually with separation of whole blood for each integral part and will comprise that leukocytic part (being commonly referred to buffy coat) is separated and cracking to discharge nucleus and/or nucleic acid.For example, in certain embodiments, this method can comprise with whole blood centrifugal (as, in the valve treatment chamber is arranged) to form plasma layer (through the upper strata of being everlasting), red corpuscle layer (through the lower floor that is everlasting) and to comprise leukocytic interfacial layer, and remove the interfacial layer (that is buffy coat) of signal portion.The buffy coat experience is further handled.

In certain embodiments, can use ordinary method from the separation of whole blood buffy coat.Then can be with buffy coat as the sample in the described method herein.

Except that above-mentioned solid phase material, the solid phase material of other type, particularly globule can be incorporated in the microfluidic device of a plurality of embodiments of the present invention.For example, can be globule is functionalized with suitable group, be used to separate specific cell, virus, bacterium, protein, nucleic acid etc.Can be by centrifugal and separating subsequently from the sample separation globule.Globule can be designed to have and be used for isolating appropriate density and size (nanometer is to micron).For example, in the situation that virus is caught, can before or after the inhibitor of from serum sample, removing small amount of residual, use the globule of the protein enclosure of identification virus to catch and make viral concentrating.

Can before or after being trapped on the globule, virus use solid phase material to remove inhibitor, as described herein.Handle outside these, can as _ _ _ title submitted to be " METHODS FOR NUCLEIC ACID ISOLATION AND KITS USING AMICROFLUIDIC DEVICE AND CONCENTRATION STEP " U.S. Patent application _ _ _ (Attorney Docket No.59801US002) in disclosed use concentrate/separate/amount of optional dilution step reduction inhibitor.This concentrating/separate/optional dilution step can be used for described several different methods and sample herein.

Can extract nucleic acid from isolating virus particle by cracking.Therefore, globule can be provided in the method that concentrates associated materials in the interior specific region of microfluidic device, also allows to wash uncorrelated material and wash-out associated materials from trapped particle.

The example of this globule includes but not limited to crosslinked polystyrene beads, it can derive from Bio-Rad Laboratories, Inc. (Hercules, CA) under the trade name CHELEX, has the crosslinked agarose beads of three (2-amino-ethyl) amine, iminodiethanoic acid, nitrilotriacetic acid(NTA), polyamine and poly-imines; And available from Rohm and Haas (Philadelphia, trade name DUOLITE C-467 PA) and the chelating ion exchange resin under the DUOLITE GT73; AMBERLITE IRC-748, DIAION CR11, DUOLITE C647.These globules also are suitable as aforesaid solid phase material.

Other example of globule comprises (the Eurobio available from trade name GENE FIZZ, France), GENE RELEASER (Bioventures Inc., Murfreesboro, TN) and BUGS NBEADS (GenPoint, Oslo, Norway) under those, and the Zymo globule (ZymoResearch, Orange is CA) with DYNAL globule (Dynal, Oslo, Norway).

Other material also can be used for pathogenic agent catch (as, virus particle, bacterium).For example, polymeric coating also can be used to separate specific cell, virus, bacterium, protein, nucleic acid etc. in certain embodiments of the invention.These polymeric coatings can be sprayed on the mulch film of microfluidic device for example by direct jet.

Can be covalently bound on little bead surface and virus particle is captured on the globule by making antibody.Antibody can be used to resist the coat protein of virus.For example, the DYNAL globule can be used for covalently bound antibody.Perhaps, synthetic polymer such as anion exchange polymer can be used for the concentrating virus particle.(Biotech Support Group, East Brunswick NJ) can be used for being coated with on globule or the select location in microfluidic device as polymeric coating, with the concentrating virus particle for commercially available resin such as viraffinity.(GenPoint, Oslo Norway) can be used for for example extraction of bacterium to BUGS N BEADS.Here, these globules can be used for catching bacterium such as staphylococcus (Staphylococcus), suis (Streptococcus), intestinal bacteria (E coli), Salmonellas (Salmonella) and Clamydia elementary body.

Therefore, in one embodiment of the invention, when sample comprises virus particle or other pathogenic agent when (as, bacterium), microfluidic device can comprise that virus catches the solid phase material of globule or other pathogenic agent capture material form.More specifically, in a kind of situation, virus is caught globule can only be used for concentrating of virus or bacterium, for example subsequently globule is separated in another chamber, and finishes with the cracking of virus or bacterium.In another kind of situation, globule can be used for concentrating of virus or bacterium, and cracking and capture nucleic acid on identical globule with the globule dilution, concentrate globule subsequently, separate globule, and repeated this process repeatedly before the nucleic acid of elute captured.

If the downstream application of nucleic acid is to make its experience amplification procedure such as PCR, all reagent that then are preferred for this method all compatible with this process (as, PCR is compatible).In addition, the adding of PCR promotor (facilitators) comes in handy, especially for diagnostic purpose.In addition, the material heating that will increase before amplification may be favourable.

Do not remove fully therein in the embodiment of inhibitor, can add damping fluid, enzyme and PCR promotor, they help the amplification procedure in the presence of inhibitor.For example, can use the enzyme that is different from the Taq polysaccharase that inhibitor is had more tolerance,, thereby provide huge benefit for pcr amplification as rTth.Can add bovine serum albumin, trimethyl-glycine, protease inhibitor, ox transferrin etc., known they can further help amplification procedure.Perhaps, (EMDBiosciences, Darmstadt Germany), are used for from directly amplification and need not large-scale purifying of whole blood can to use commercially available product such as Novagen ' s Blood Direct PCR Buffer test kit.

Further specify objects and advantages of the present invention by following examples, but the concrete material narrated in these embodiments and amount thereof and other condition and details should be interpreted as and exceedingly limit the present invention.

Embodiment

Embodiment 1A: the preparation of solid phase material: the ammonia form that does not have TRITON X-100

3M No.2271 EMPORE Extraction chelating is placed the glass filter support with dish.To extract the form that dish is converted into ammonia according to the method that is printed on the package insert.

Embodiment 1B: the preparation of solid phase material: ammonia form with TRITON X-100

3M No.2271 EMPORE Extraction chelating is placed the glass filter support with dish.To extract the form that dish is converted into ammonia according to the method that is printed on the package insert.Dish is placed bottle and be immersed in 1%TRITON X-100 (Sigma-Aldrich, St.Louis, MO) in the solution (the TRITON X-100 of 0.1 gram (g) is in 10mL water), at Thermolyne Vari-Mix Model M48725 Rocker (Barnstead/Thermolyne, Dubuque IA) goes up mixing about 6-8 hour.Dish is placed on the glass filter support, and by applying about 20 minutes (min) dryings of vacuum, dried overnight under room temperature (about 21 ℃) is noted not washing and rinsing then.

Embodiment 1C: the preparation of solid phase material: Anion-SREmpore chelating membrane with TRITON X-100

3M No.2252 EMPORE Extraction chelating is placed bottle with dish and be immersed in 1%TRITON X-100 solution (the TRITON X-100 of 0.1g is at 10 milliliters of (mL) water), on Thermolyne Vari-Mix Model M48725 Rocker, mixed about 6-8 hour.Dish is placed on the glass filter support, and by applying about 20 minutes (min) dryings of vacuum, dried overnight under room temperature (about 21 ℃) is noted not washing and rinsing then.

Embodiment 1D: the preparation of solid phase material: C8 chelating membrane with TRITON X-100

3M No.2214 EMPORE Extraction chelating is placed bottle with dish and be immersed in 1%TRITON X-100 solution (the TRITON X-100 of 0.1g is at 10 milliliters of (mL) water), on Thermolyne Vari-Mix Model M48725 Rocker, mixed about 6-8 hour.Dish is placed on the glass filter support, and by applying about 20 minutes (min) dryings of vacuum, dried overnight under room temperature (about 21 ℃) is noted not washing and rinsing then.

Embodiment 1E: the preparation of solid phase material: chelating membrane with TRITON X-100 and FR-2025

3M No.2271 EMPORE Extraction chelating is placed bottle with dish and be immersed in 1%TRITON X-100 solution (the TRITON X-100 of 0.1g is at 10 milliliters of (mL) water) and 0.1%FR-2025 (3M, St.Paul, MN) be in 50/50 the mixture, and on Thermolyne Vari-Mix Model M48725 Rocker, mixed about 6-8 hour.Dish is placed on the glass filter support, and by applying about 20 minutes (min) dryings of vacuum, dried overnight under room temperature (about 21 ℃) is noted not washing and rinsing then.

Embodiment 1F: the preparation of solid phase material: chelating membrane with TRITON X-100 and FC-4430

3M No.2271 EMPORE Extraction chelating is placed bottle with dish and be suspended in 1%TRITON X-100 (the TRITON X-100 of 0.1g is at 10 milliliters of (mL) water) and 0.1%FC-4430 (3M, St.Paul, MN) be in 50/50 the mixture, and on Thermolyne Vari-Mix Model M48725 Rocker, mixed about 6-8 hour.Dish is placed on the glass filter support, and by applying about 20 minutes (min) dryings of vacuum, dried overnight under room temperature (about 21 ℃) is noted not washing and rinsing then.

Embodiment 1G: the preparation of solid phase material: chelating membrane with TRITON X-100 and PSSA

3M No.2271 EMPORE Extraction chelating is placed the glass filter support with dish.Should extract according to the method for printing on the package insert and to be converted into the ammonia shape with dish.Dish is placed bottle and be immersed in 1%TRITON X-100 (TRITONX-100 of 0.1g is at 10 milliliters of (mL) water) and 1%PSSA (poly-(4-Sodium styrene sulfonate) (Sigma-Aldrich, St.Louis, MO) (20 weight %PSSA storing solutions are diluted to 1 weight % in the sterilized water of qiagen rnase enzyme not) and are in 50/50 the mixture, and on ThermolyneVari-Mix Model M48725 Rocker, mixed about 6-8 hour.Dish is placed on the glass filter support, and by applying about 20 minutes (min) dryings of vacuum, dried overnight under room temperature (about 21 ℃) is noted not washing and rinsing then.

Embodiment 1H: the preparation of solid phase material: Cation-SREMPORE chelating membrane with TRITON X-100

3M No.2251 EMPORE Extraction chelating is placed bottle with dish and be immersed in 1%TRITON X-100 solution (the TRITON X-100 of 0.1g is at 10mL water), on Thermolyne Vari-Mix Model M48725 Rocker, mixed about 6-8 hour.Dish is placed on the glass filter support, and by applying about 20 minutes (min) dryings of vacuum, dried overnight under room temperature (about 21 ℃) is noted not washing and rinsing then.

Embodiment 1J: the preparation of solid phase material: EMPORE chelating membrane with TRITON X-100

3M No.2241 EMPORE Extraction chelating is placed bottle with dish and be immersed in 1%TRITON X-100 solution (the TRITON X-100 of 0.1g is at 10mL water), on Thermolyne Vari-Mix Model M48725 Rocker, mixed about 6-8 hour.Dish is placed on the glass filter support, and by applying about 20 minutes (min) dryings of vacuum, dried overnight under room temperature (about 21 ℃) is noted not washing and rinsing then.

Embodiment 2: use the method for chelating solid phase material from the separation of whole blood genomic dna

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution Eppendorf Model 5415D whizzer (Brinkmann, Westbury, NY) at 400 rcf centrifugal about 10 minutes.Separate and abandoning supernatant, stay the condensed matter of about two (2) μ L of centrifuge tube bottom.This material transfer to 3M No.2271 EMPORE Extraction chelating dish, is guaranteed that material is evenly distributed on the surface of dish.Allow material on dish dry about 2-5 minute, deepen up to the intensive redness.To extracting the NaOH that adds 0.083 mole (M) of 12 microlitres (12 μ L) with dish.Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is for from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and (Sigma-Aldrich, St.Louis is MO) in (pH 7.4) with its TRIS-HCl that joins 40 mmoles (mM) of 10 μ L.

Embodiment 3: use the method for the chelating solid phase material of ammonification from the separation of whole blood genomic dna

The pure TRITON X-100 of one (1) μ L joins in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Separate and abandoning supernatant, stay the condensed matter of about two (2) μ L of centrifuge tube bottom.This substance transfer to the extraction film for preparing, is guaranteed that material is evenly distributed on the surface of film in embodiment 1A.Allow material on film dry about 2-5 minute, deepen up to the intensive redness.To extracting the NaOH that film adds 0.083 mole (M) of 12 microlitres (12 μ L).Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and (Sigma-Aldrich, St.Louis is MO) in (pH 7.4) with its TRIS-HCl that joins 40 mmoles (mM) of 10 μ L.

Embodiment 4: use the method for the chelating solid phase material of the ammonification with TRITON X-100 from the separation of whole blood genomic dna

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Separate and abandoning supernatant, stay the condensed matter of about two (2) μ L of centrifuge tube bottom.This substance transfer to the extraction film for preparing, is guaranteed that material is evenly distributed on the surface of film in embodiment 1B.Allow material on film dry about 2-5 minute, deepen up to the intensive redness.To extracting the NaOH that film adds 0.083 mole (M) of 12 microlitres (12 μ L).Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and (Sigma-Aldrich, St.Louis is MO) in (pH 7.4) with its TRIS-HCl that joins 40 mmoles (mM) of 10 μ L.

Embodiment 5A: before handling with solid phase material by whole blood is carried out centrifugal and concentrated genomic dna: from pipe top sample separation

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Take out the aliquots containig of two (2) μ L and transfer to the extraction film that among embodiment 1B, prepares from the top of centrifuge tube, guarantee that material is evenly distributed on the surface of film.Allow material on film dry about 2-5 minute, deepen up to the intensive redness.To extracting the NaOH that film adds 0.083 mole (M) of 12 microlitres (12 μ L).Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of 40 mmoles (mM) of 10 μ L.

Embodiment 5B: before handling with solid phase material by whole blood is carried out centrifugal and concentrated genomic dna: from pipe bottom sample separation

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Take out the aliquots containig of two (2) μ L and transfer to the extraction film that among embodiment 1B, prepares from the bottom of centrifuge tube, guarantee that material is evenly distributed on the surface of film.Allow material on film dry about 2-5 minute, deepen up to the intensive redness.To extracting the NaOH that film adds 0.083 mole (M) of 12 microlitres (12 μ L).Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of 40 mmoles (mM) of 10 μ L.

Embodiment 5C: by using the processing purified genomic dna of solid phase material: from the top sample separation of pipe-do not have enrichment step

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.Take out the aliquots containig of two (2) μ L and transfer to the extraction film that among embodiment 1B, prepares from the top of centrifuge tube, guarantee that material is evenly distributed on the surface of film.Allow material on film dry about 2-5 minute, deepen up to the intensive redness.To extracting the NaOH that film adds 0.083 mole (M) of 12 microlitres (12 μ L).Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of 40 mmoles (mM) of 10 μ L.

Embodiment 6A: by the centrifugal QIAamp that carries out subsequently of whole blood purify is concentrated genomic dna: from pipe top sample separation

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Take out the aliquots containig of two (2) μ L from the top of centrifuge tube and join the phosphate buffered saline (PBS) (PBS) of 198 μ L.According at QIAamp DNA Blood Mini Kit Handbook, the 27th page (CA) described in " Blood and Body Fluid Spin Protocol " obtains clean DNA with the water elution of 72 μ L for Qiagen, Valencia.

Embodiment 6B: by the centrifugal QIAamp that carries out subsequently of whole blood purify is concentrated genomic dna: from pipe bottom sample separation

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Take out the aliquots containig of two (2) μ L from the bottom of centrifuge tube and join the 1X phosphate buffered saline (PBS) (PBS) of 198 μ L.According at QIAamp DNA Bloodmini Kit Handbook, the 27th page (CA) described in " Blood and Body Fluid Spin Protocol " obtains clean DNA with the water elution of 72 μ L for Qiagen, Valencia.

Embodiment 6C: collect genomic dna: from managing top sample separation one without centrifugal by the QIAamp purification

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.Take out the aliquots containig of two (2) μ L from the top of centrifuge tube and join the 1X phosphate buffered saline (PBS) (PB S) of 198 μ L.According at QIAamp DNA Bloodmini Kit Handbook, the 27th page (CA) described in " Blood and Body Fluid Spin Protocol " obtains clean DNA with the water elution of 72 μ L for Qiagen, Valencia.

Embodiment 7A: inhibitor/DNA is to the influence of PCR: change inhibitor concentration under fixed DNA concentration

Before strengthening pure human gene group DNA, make the serial dilution thing of inhibitor, so that the research inhibitor is to the influence of PCR.(sample 7-does not add inhibitor to add the different Mix I (pure or its dilution) of 1 μ L in 15 nanograms/microlitre (ng/ μ L) human gene group DNA of 10 μ L, 300) and vortex 7D-is pure, 7E-1: 10,7F-1: 30,7G-1: 100,7H-1:.Two (2) the μ L aliquot sample that obtain each sample are used for the PCR of 20 μ L.The result is as shown in table 2.

Mix I: the pure TRITON X-100 that in the whole blood of 100 (100) μ L, adds 1 μ L.Solution was cultivated about 5 minutes down in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it is transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer centrifugal about 10 minutes at 400rcf.Obtain about 80 μ L from the top of Eppendorf tube and be appointed as Mix I.

Embodiment 7B: inhibitor/DNA is to the influence of PCR: change DNA concentration under the fixed inhibitor concentration

The Mix I (aforesaid) that in the human gene group DNA of 10 μ L, adds the dilution in 1: 3 of 1 μ L.The DNA concentration of research is as follows: sample 7J-15ng/ μ L, 7K-7.5ng/ μ L, 7L-3.75ng/ μ L, 7M-1.5ng/ μ L.The aliquot sample that obtains two (2) μ L from each sample is used for the PCR of 20 μ L.The result is as shown in table 2.

Embodiment 7C: inhibitor/DNA is to the influence of PCR: the DNA that does not add inhibitor

The water that adds 1 μ L in each DNA sample replaces the following sample of inhibitor preparation: sample 7N-15ng/ μ L, 7P-7.5ng/ μ L, 7Q-3.75ng/ μ L, 7R-1.5ng/ μ L.The aliquot sample that obtains two (2) μ L from each sample is used for the PCR of 20 μ L.The result is as shown in table 2.

Table 2

Sample number into spectrum	Ct (duplicate sample)	Sample number into spectrum	Ct (duplicate sample)
				7	19.10 19.06	7K	29.16 30.22
7D	13.94 29.50	7L	30.47 29.96
				7E	27.39 26.22	7M	28.43 26.16
7F	21.44 20.66	7N	20.05 19.80
				7G	19.90 19.30	7P	20.74 20.54
7H	19.90 20.08	7Q	21.95 21.88
				7J	28.45 28.61	7R	22.67 23.10

Embodiment 8A: reclaim nucleus from solid phase material

The pure TRITON X-100 of one (1) μ L is joined in the white corpuscle (PBMC) of 2 μ L.With solution vortex and in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 1 minute momently.Three (3) μ L are placed on the chelating membrane for preparing among the embodiment 1B.Allow material on film dry about 2-5 minute.To extracting the 0.077MNaOH that film adds ten three (13) μ L.Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 13 μ L).If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of 40 mmoles (mM) of 10 μ L.

Embodiment 8B: do not use solid phase material to reclaim nucleus

The pure TRITON X-100 of one (1) μ L is joined in the white corpuscle (PBMC) of 2 μ L.With solution vortex and in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 1 minute momently.In solution, add the 0.1M NaOH of ten (10) μ L and cultivated about 5 minutes in room temperature (about 21 ℃).Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of the 40mM of 10 μ L.

Embodiment 8C: the sample of strengthening with nucleus afterwards

The pure TRITON X-100 of one (1) μ L is joined in the 2 μ L water.With solution vortex and in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 1 minute momently.Three (3) μ L are placed on the chelating membrane for preparing among the embodiment 1B.Allow material on film dry about 2-5 minute.To extracting the 0.077M NaOH that film adds ten three (13) μ L.Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 13 μ L).If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.The white corpuscle (PBMC) of two (2) μ L is joined in the 10 μ L aliquots containigs of solution and and cultivated about 5 minutes in room temperature (about 21 ℃).Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of the 40mM of 10 μ L.

Embodiment 9A: use the method for solid phase material: extract from solid phase material with NaOH from the separation of whole blood genomic dna

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Separate and abandoning supernatant, stay the condensed matter of about two (2) μ L of centrifuge tube bottom.This substance transfer to the extraction film for preparing, is guaranteed that material is evenly distributed on the surface of film in embodiment 1B.Allow material on film dry about 2-5 minute, deepen up to the intensive redness.To extracting the NaOH that film adds 0.083 mole (M) of 12 microlitres (12 μ L).Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.To 10 μ L aliquots containigs of solution add 50 (50) μ L 1X TE buffer reagent (Sigma-Aldrich, St.Louis, MO).The TRIS-HCl (pH 7.4) that in solution, adds the 40mM of 300 (300) μ L.

Embodiment 9B: use the method for solid phase material from the separation of whole blood genomic dna: water extracts from solid phase material

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Separate and abandoning supernatant, stay the condensed matter of about two (2) μ L of centrifuge tube bottom.This substance transfer to the extraction film for preparing, is guaranteed that material is evenly distributed on the surface of film in embodiment 1B.Allow material on film dry about 2-5 minute, deepen up to the intensive redness.To extracting the water that film adds 12 microlitres (12 μ L).Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Add the 0.1M NaOH of 50 (50) μ L and cultivated about 5 minutes to 10 μ L aliquots containigs of solution in room temperature (about 21 ℃).The TRIS-HCl (pH7.4) that in solution, adds the 40mM of 300 (300) μ L.

Embodiment 10: on the chelating solid phase material of the ammonification of handling with TRITON X-100 from the separation of whole blood genomic dna

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.This substance transfer to the extraction film for preparing, is guaranteed that material is evenly distributed on the surface of film in embodiment 1B.Allow material on film dry about 2-5 minute, deepen up to the intensive redness.To extracting the 0.083M NaOH that film adds ten three (13) μ L.Solution after mixing 2-3 time up and down and mixing, is removed the transfer pipet end.Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of the 40mM of 10 μ L.

Embodiment 11: use the method for Anion-SR chelating solid phase material from the separation of whole blood genomic dna

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Separate and abandoning supernatant, stay about two (2) mL condensed matters of centrifuge tube bottom.With this substance transfer to the 3M No.2252 EMPORE Anion-SRExtraction chelating that described in embodiment 1C, prepares with dish on, guarantee that material is evenly distributed on the surface of dish.Allow material on dish dry about 2-5 minute, deepen up to the intensive redness.To extracting the 0.083M NaOH that adds ten two (12) μ L with dish.Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of the 40mM of 10 μ L.

Embodiment 12: use the method for C8 chelating solid phase material from the separation of whole blood genomic dna

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Separate and abandoning supernatant, stay about two (2) mL condensed matters of centrifuge tube bottom.With this substance transfer to the 3M No.2214 EMPORE C-8 Extraction chelating that described in embodiment 1D, prepares with dish on, guarantee that material is evenly distributed on the surface of dish.Allow material on dish dry about 2-5 minute, deepen up to the intensive redness.To extracting the 0.083M NaOH that dish adds ten two (12) μ L.Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of the 40mM of 10 μ L.

Embodiment 13: use the method for chelating solid phase material from the separation of whole blood genomic dna

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Separate and abandoning supernatant, stay the condensed matter of about two (2) μ L of centrifuge tube bottom.With this substance transfer to the 3M No.2271 EMPORE Extraction chelating that described in embodiment 1E, prepares with dish on, guarantee that material is evenly distributed on the surface of dish.Allow material on dish dry about 2-5 minute, deepen up to the intensive redness.To extracting the 0.083M NaOH that dish adds ten two (12) μ L.Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of the 40mM of 10 μ L.

Embodiment 14: use the method for chelating solid phase material from the separation of whole blood genomic dna

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Separate and abandoning supernatant, stay the condensed matter of about two (2) μ L of centrifuge tube bottom.With this substance transfer to the 3M No.2271 EMPORE Extraction chelating that described in embodiment 1F, prepares with dish on, guarantee that material is evenly distributed on the surface of dish.Allow material on dish dry about 2-5 minute, deepen up to the intensive redness.To extracting the O.083M NaOH that dish adds ten two (12) μ L.Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of the 40mM of 10 μ L.

Embodiment 15: use the method for chelating solid phase material from the separation of whole blood genomic dna

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Separate and abandoning supernatant, stay about two (2) mL condensed matters of centrifuge tube bottom.With this substance transfer to the 3M No.2271EMPORE Extraction chelating that described in embodiment 1G, prepares with dish on, guarantee that material is evenly distributed on the surface of dish.Allow material on dish dry about 2-5 minute, deepen up to the intensive redness.To extracting the 0.083M NaOH that dish adds ten two (12) μ L.Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of the 40mM of 10 μ L.

Embodiment 16: use the method for chelating solid phase material from the separation of whole blood genomic dna

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Separate and abandoning supernatant, stay the condensed matter of about two (2) μ L of centrifuge tube bottom.With this substance transfer to the 3M No.2271 EMPORE Extraction chelating that described in embodiment 1H, prepares with dish on, guarantee that material is evenly distributed on the surface of dish.Allow material on dish dry about 2-5 minute, deepen up to the intensive redness.To extracting the 0.083M NaOH that dish adds ten two (12) μ L.Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of the 40mM of 10 μ L.

Embodiment 17: use the method for chelating solid phase material from the separation of whole blood genomic dna

The pure TRITON X-100 of one (1) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 10 minutes.Separate and abandoning supernatant, stay the condensed matter of about two (2) μ L of centrifuge tube bottom.With this substance transfer to the 3M No.2271 EMPORE Extraction chelating that described in embodiment 1J, prepares with dish on, guarantee that material is evenly distributed on the surface of dish.Allow material on dish dry about 2-5 minute, deepen up to the intensive redness.To extracting the 0.083M NaOH that dish adds ten two (12) μ L.Solution after mixing 2-3 time up and down and mixing, the transfer pipet end is removed (though may obtain being less than 12 μ L).The color of the sample of removing from film is from the colourless light green that becomes.If the solution show bubble is then with its at 4,000 rev/mins (rpm) centrifugal 1 minute.Take out the aliquots containig of 2 μ L and it is joined among the TRIS-HCl (pH 7.4) of the 40mM of 10 μ L.

Embodiment 18: use the method for the chelating solid phase material of the ammonification with TRITON X-100 on the microfluidic device from the separation of whole blood genomic dna that be installed in

The pure TRITON X-100 of two (2) μ L is joined in the whole blood of 100 (100) μ L.Solution was cultivated about 5 minutes in room temperature (about 21 ℃), with solution eddy current (carrying out about 5 seconds in per 20 seconds) off and on.Observe solution to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 2 minutes.Top solution is abandoned, stayed 4 μ L of pipe bottom.In the concentrated material of 4 μ L, add the rnase water of 4 μ L and with the sample thorough mixing.Then sample is placed in the injection port on the microfluidic device (Fig. 1) described in the U.S. Patent application of submitting on December 12nd, 2003 as applicant's transferee co-pending 10/734,682.Solution comprised the 3M No.2271 EMPORE Extraction chelating that described in embodiment 1B, prepares chamber centrifugal 5 minutes of 500rpm with cleaning, use 10%X-100 to replace 1%TRITON X-100 as reinforced solution with dish.Join five (5) the μ L aliquots containigs of 0.1MNaOH in the different injection ports and centrifugal 5 minutes, be used for as mentioned above the chamber being cleaned at 1000rpm.Ten (10) the μ L aliquots containigs of 0.1M NaOH are joined in the identical injection port and with solution centrifugal 10 minutes at 2500rpm.Take out 5 μ L and it is joined among the TRIS-HCl (pH 7.4) of the 1M of 1.5 μ L.

Embodiment 19: use the method for chelating solid phase material from the separation of whole blood genomic dna

The whole blood of 100 (100) μ L is joined among the 2%TRITON X-100 of 100 (100) μ L.The solution thorough mixing is observed solution then to be sure of that it was transparent before carrying out next step.With solution in Eppendorf Model 5415D whizzer under 400rcf centrifugal about 2 minutes.Remove 95 μ L aliquots containigs of solution from the top and abandon.Last five (5) the μ L that will comprise concentrated material place the 3M No.2271 EMPOREExtraction chelating for preparing described in embodiment 1B with on the dish, use 10%TRITON X-100 to replace 1%TRITONX-100 as reinforced solution.After solution is immersed in the dish, sample is extracted with 20 (20) the μ L aliquots containigs of 0.1M NaOH.Solution is centrifugal momently under 400rcf in Eppendorf Model 5415D whizzer.Ten one (11) μ L aliquots containigs of sample were heated 3 minutes at 95 ℃, join then among the 1M TRIS-HCl (pH7.4) of three (3) μ L.

Embodiment 20: from the method for separation of whole blood genomic dna

The whole blood of five (5) μ L is joined among the 10mM NaOH of ten (10) μ L.After cultivating 1 minute, sample was heated 3 minutes at 95 ℃.Five (5) the μ L aliquots containigs of 16mM TRIS-HCl (pH 7.4) are joined in the sample of 10 μ L.

The result

Table 3 reported according in the QuantiTech SYBR Green PCR Handbook 10-12 page or leaf about preparing the guidance of 10 μ L PCR samples (2 μ L samples in 10 μ L SYBR Green Master Mix, the beta-actin of 4 μ L, the water of 4 μ L), at ABI 7700 QPCRMachine (Applera, Foster City, the embodiment 1-17 that obtains on CA), 19 and 20 result; The result of embodiment 18 according to about the package insert 2-3 page or leaf of the LightCycler Factor V Leiden Mutation test kit for preparing 10 μ L PCR samples (2.5 μ L samples, the 10x Factor V Leiden ReactionMix of 1 μ L and the 10x Factor V Leiden Mutation Detection Mix of 1 μ L in the sterilized water of the not qiagen rnase enzyme of 5.5 μ L) at LightCycler 2.0 (Roche Applied Science, Indianapolis obtains on IN).Horizon 11-14 Electrophoresis Machine (Gibco BRL, G aithersburg, MD) go up operation 1% sepharose (bands of a spectrum brightness-+fuzzy, +++bright).Under 405nm at SpectraMax Plus ³⁸⁴(Molecular Devices Corporation, Sunnyvale CA.) carry out spectral measurement on the spectrophotometer.Two, three of each sample or four numeric representations are duplicate, in triplicate or quadruplicate.

Table 3

Sample	Ct	Bands of a spectrum	405nm (mean value)
				1.5ng/ μ L human gene group DNA in 0.1M NaOH/40mM TRIS-HCl damping fluid	16.92 20.67	+++ +++	-
1.5ng/ μ L human gene group DNA in water	19.01 18.67	+++ +++	0
				1.5ng/ μ L human gene group DNA in water	16.18 16.28	+++ +++	-
Embodiment 4	17-20 (deriving from analysis) to a plurality of samples	+++ +++	0.24
				Embodiment 3	26、28	+	-
Embodiment 2	28、27	Do not have	-
				Embodiment 5A	28 28	+ +	0.37
Embodiment 5B	17、18	+++ +++	0.24

Embodiment 5C	24 26	++ ++	-
				Embodiment 6A	23 24	+ +	0
Embodiment 6B	17 18	+++ +++	0
				Embodiment 6C	21 21	++ ++	0
The Mix I of embodiment 7A and 7B dilution in 1: 36	-	-	2.63
				The Mix I of embodiment 7A and 7B dilution in 1: 360	-	-	0.38
The Mix I of embodiment 7A and 7B dilution in 1: 3600	-	-	0.036
				The Mix I of embodiment 7A and 7B dilution in 1: 36000	-	-	0
Embodiment 8A	22 21 18 22	+++	-
				Embodiment 8B	22 21 18 21	++	-
Embodiment 8C	20 20 21 22	-	-
				Embodiment 9A	21 19	-	0.019 0.007

Embodiment 9B	25 28	-	0.029 0.074
				Embodiment 10	26.86 26.11	+++	-
Embodiment 11	24.87 20.38	-	-
				Embodiment 12	26.74 26.04	-	-
Embodiment 13	26.01	-	0.245
				Embodiment 14	19.82	-	0.377
Embodiment 15	27.92 30.04
				Embodiment 16	-	-	0.07
Embodiment 17	-	-	0.918
				Embodiment 18	(27.39 worktable contrast)	-	-
Embodiment 19 *	23.72、24.03	-	-
				Embodiment 20 *	30.35、30.56	-	-

^*The DNA that the positive control of embodiment 19-20 extracts from 200 (200) μ L whole bloods according to " Blood and Body Fluid Spin Protocol " described in the 27th page of the QIAamp DNA Blood Mini KitHandbook, wash-out in 200 μ L water, and have 23 Ct value.In these experiments, negative control (NTC or do not have template contrast) is not increased.

Multiple change of the present invention and variation will become apparent to those skilled in the art that and do not depart from the scope of the present invention.Should be appreciated that the present invention is not subjected to exemplary that this paper proposes and the excessive restriction of embodiment, and this embodiment and embodiment exist as an example just, scope of the present invention is only by the following claim restriction that proposes herein.

Claims (47)

1. from the method for sample separation nucleic acid, this method comprises:

Provide and comprise the material that contains nucleic acid and the sample of inhibitor;

Form the sample concentration district; Wherein the sample concentration district comprises material and the inhibitor that contains nucleic acid;

The sample concentration district is separated basically with sample time enrichment region;

Make isolating sample concentration district contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration;

Before making isolating sample concentration district contact solid phase material, simultaneously or afterwards randomly cracking contains the material of nucleic acid to discharge nucleic acid; With

The material and/or the nucleic acid that make at least a portion contain nucleic acid separate with the solid phase material that has adhered at least a portion inhibitor.

2. from the method for sample separation nucleic acid, this method comprises:

The sample that comprises the material that contains nucleic acid and contain the cell of inhibitor is provided;

Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor;

Form the lysate sample enrichment region; Wherein the lysate sample enrichment region comprises material and the inhibitor that contains nucleic acid;

The lysate sample enrichment region is separated basically with lysate sample time enrichment region;

Make isolating lysate sample enrichment region contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration;

Before making isolating lysate sample enrichment region contact solid phase material, simultaneously or afterwards randomly further cracking contain the material of nucleic acid to discharge nucleic acid; With

The material and/or the nucleic acid that make at least a portion contain nucleic acid separate with the solid phase material that has adhered at least a portion inhibitor.

3. from the method for sample separation nucleic acid, this method comprises:

Provide and comprise cell that contains inhibitor and the sample that contains nuclear cell;

Make biological sample effectively destroy cytolemma and discharge nucleus and inhibitor and form under the condition of lysate sample and contact nonionic surface active agent;

Form the lysate sample enrichment region; Wherein the sample concentration district comprises nucleus and inhibitor;

The lysate sample enrichment region is separated basically with lysate sample time enrichment region;

Make isolating lysate sample enrichment region contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration;

Before making isolating lysate sample enrichment region contact solid phase material, simultaneously or afterwards randomly further lysing cell nuclear to discharge nucleic acid; With

At least a portion nucleus and/or nucleic acid are separated with the solid phase material that has adhered at least a portion inhibitor.

4. from the method for sample separation nucleic acid, this method comprises:

Microfluidic device is provided, and this microfluidic device comprises feed compartment, the valve treatment chamber is arranged and comprise the separate chamber of solid phase material;

Provide and comprise the material that contains nucleic acid and the sample of inhibitor;

Sample is placed feed compartment;

With sample transfer to the valve treatment chamber is arranged;

Form the sample concentration district in the valve treatment chamber is arranged, wherein the sample concentration district comprises material and the inhibitor that contains nucleic acid;

The sample concentration district is separated basically with sample time enrichment region;

Isolating sample concentration district is transferred to the separate chamber contact, make at least a portion inhibitor preferentially be attached to solid phase material with solid phase material; Wherein solid phase material comprises and catches the position, is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration;

Before making isolating sample concentration district contact solid phase material, simultaneously or afterwards randomly cracking contains the material of nucleic acid to discharge nucleic acid; With

The material and/or the nucleic acid that make at least a portion contain nucleic acid separate with the solid phase material that has adhered at least a portion inhibitor.

5. from the method for sample separation nucleic acid, this method comprises:

Microfluidic device is provided, and this microfluidic device comprises feed compartment, the valve treatment chamber is arranged and comprise the separate chamber of solid phase material;

The sample that comprises the material that contains nucleic acid and contain the cell of inhibitor is provided;

Sample is placed feed compartment;

Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor;

Lysate sample has been transferred to the valve treatment chamber;

Form the lysate sample enrichment region in the valve treatment chamber is arranged, wherein the lysate sample enrichment region comprises material and the inhibitor that contains nucleic acid;

The lysate sample enrichment region is separated basically with lysate sample time enrichment region;

Isolating lysate sample enrichment region is transferred to the separate chamber contact, make at least a portion inhibitor preferentially be attached to solid phase material with solid phase material; Wherein solid phase material comprises and catches the position, is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration;

Before making isolating lysate sample enrichment region contact solid phase material, simultaneously or afterwards randomly further cracking contain the material of nucleic acid to discharge nucleic acid; With

The material and/or the nucleic acid that make at least a portion contain nucleic acid separate with the solid phase material that has adhered at least a portion inhibitor.

6. the method for claim 5, the material that wherein contains nucleic acid comprise that nucleus and this method comprise makes at least a portion nucleus separate with solid phase material.

7. the method for claim 5 wherein makes at least a portion contain the material of nucleic acid and/or nucleic acid and separates the material and/or the nucleic acid that comprise containing nucleic acid with solid phase material and transfer to the amplified reaction chamber.

8. the method for claim 7, it further comprises makes nucleus and/or nucleic acid experience amplified reaction.

9. the method for claim 5, wherein making at least a portion contain the material of nucleic acid and/or nucleic acid separates with solid phase material and comprises material and/or the nucleic acid that contains nucleic acid with eluent from solid phase material wash-out at least a portion, randomly heating contains the material and/or the nucleus of nucleic acid and randomly regulates pH.

10. from the method for sample separation nucleic acid, this method comprises:

Microfluidic device is provided, and this microfluidic device comprises feed compartment, the valve treatment chamber is arranged and comprise the separate chamber of solid phase material;

Provide and comprise cell that contains inhibitor and the sample that contains nuclear cell;

Sample is placed feed compartment;

Make sample contact nonionic surface active agent under with the condition that forms lysate sample effectively destroying cytolemma and discharge nucleus and inhibitor;

Lysate sample has been transferred to the valve treatment chamber;

Form the lysate sample enrichment region in the valve treatment chamber is arranged, wherein the lysate sample enrichment region comprises nucleus and inhibitor;

The lysate sample enrichment region is separated basically with lysate sample time enrichment region;

Isolating lysate sample enrichment region is transferred to the separate chamber contact, make at least a portion inhibitor preferentially be attached to solid phase material with solid phase material; Wherein solid phase material comprises and catches the position, is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration;

Before making isolating lysate sample enrichment region contact solid phase material, simultaneously or afterwards randomly further lysing cell nuclear to discharge nucleic acid; With

At least a portion nucleus and/or nucleic acid are separated with the solid phase material that has adhered at least a portion inhibitor.

11. the method for claim 10 wherein makes at least a portion nucleus and/or nucleic acid separate with solid phase material to comprise nucleus and/or nucleic acid is transferred to the amplified reaction chamber.

12. the method for claim 11, it further comprises and randomly heats nucleus and/or nucleic acid, randomly regulates pH and make nucleus and/or nucleic acid experience amplified reaction.

13. the method for claim 10 wherein places sample feed compartment to occur in before the sample contact nonionic surface active agent.

14. the method for claim 10, wherein feed compartment comprises that the nonionic surface active agent that presets and sample contact nonionic surface active agent occur in when sample placed feed compartment.

15. the method for claim 10 wherein places sample feed compartment to occur in after the sample contact nonionic surface active agent.

16. the method for claim 10, wherein sample contact nonionic surface active agent in mixing section at thorough mixing to destroy cytolemma and to discharge nucleus and inhibitor carries out under with the condition that forms lysate sample.

17. the method for claim 10 wherein forms the lysate sample enrichment region and comprises and carry out centrifugal to the sample in the treatment chamber in the valve treatment chamber is arranged.

18. the method for claim 17 wherein makes lysate sample enrichment region and lysate sample time enrichment region separate to comprise by valve lysate sample time enrichment region is transferred to waste chamber.

19. the method for claim 10, wherein coating reagent comprises tensio-active agent.

20. the method for claim 10 wherein makes at least a portion nucleus and/or nucleic acid separate with solid phase material and comprises with eluent from solid phase material wash-out at least a portion nucleus and/or nucleic acid.

21. the method for claim 20, wherein eluent is a lytic reagent.

22. the method for claim 10 wherein uses second lytic reagent to carry out other cleavage step.

23. from the method for sample separation nucleic acid, this method comprises:

The sample that comprises nucleic acid and inhibitor is provided;

Make sample contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration;

If before making lysate sample contact solid phase material, simultaneously or afterwards randomly the material that contains nucleic acid that exists of cracking to discharge nucleic acid; With

Separate with the solid phase material that has adhered at least a portion inhibitor by material and/or the nucleic acid that makes at least a portion contain nucleic acid with the eluent wash-out, condition is that eluent is not a nonionic surface active agent.

24. from the method for sample separation nucleic acid, this method comprises:

The sample that comprises the material that contains nucleic acid and contain the cell of inhibitor is provided;

Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor;

Make lysate sample contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration;

Before making lysate sample contact solid phase material, simultaneously or afterwards randomly further cracking contain the material of nucleic acid to discharge nucleic acid; With

Separate with the solid phase material that has adhered at least a portion inhibitor by material and/or the nucleic acid that makes at least a portion contain nucleic acid with the eluent wash-out, condition is that eluent is not a nonionic surface active agent.

25. from the method for sample separation nucleic acid, this method comprises:

The sample that comprises the material that contains nucleic acid and contain the cell of inhibitor is provided;

Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor;

Make lysate sample contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration;

The material that makes at least a portion contain nucleic acid separates with the solid phase material that has adhered at least a portion inhibitor; With

After separation contained the material of nucleic acid, further cracking contained the material of nucleic acid to discharge nucleic acid.

26. the method for claim 25, the material that wherein contains nucleic acid comprises nucleus.

27. the method for claim 25, wherein coating reagent comprises tensio-active agent.

28. from the method for sample separation nucleic acid, this method comprises:

Microfluidic device is provided, and this microfluidic device comprises feed compartment and comprises the separate chamber of solid phase material;

The sample that comprises the material that contains nucleic acid and contain the cell of inhibitor is provided;

Sample is placed feed compartment;

Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor;

Lysate sample is transferred to the separate chamber with the contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material;

Before making sample contact solid phase material, simultaneously or afterwards randomly further cracking contain the material of nucleic acid to discharge nucleic acid; Wherein solid phase material comprises and catches the position, is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; With

Separate with the solid phase material that has adhered at least a portion inhibitor by material and/or the nucleic acid that makes at least a portion contain nucleic acid with the eluent wash-out, condition is that eluent is not a nonionic surface active agent.

29. from the method for sample separation nucleic acid, this method comprises:

Microfluidic device is provided, and this microfluidic device comprises feed compartment and comprises the separate chamber of solid phase material;

The sample that comprises the material that contains nucleic acid and contain the cell of inhibitor is provided;

Sample is placed feed compartment;

Make sample contact first lytic reagent effectively destroying under the condition of lysate sample that cytolemma and release inhibitor and formation comprises the material that contains nucleic acid and inhibitor;

Lysate sample is transferred to the separate chamber with the contact solid phase material, make at least a portion inhibitor preferentially be attached to solid phase material; Wherein solid phase material comprises and catches the position, is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; With

The material that makes at least a portion contain nucleic acid separates with the solid phase material that has adhered at least a portion inhibitor; With

After separation contained the material of nucleic acid, further cracking contained the material of nucleic acid to discharge nucleic acid.

30. the method for claim 29, the material that wherein contains nucleic acid comprises nucleus.

31. the method for claim 29 wherein makes the material that contains nucleic acid and/or nucleic acid separate the material and/or the nucleic acid that comprise containing nucleic acid with solid phase material and transfers to the amplified reaction chamber.

32. the method for claim 31, it further comprises makes material and/or the nucleic acid experience amplified reaction that contains nucleic acid.

33. the method for claim 29 wherein places sample feed compartment to occur in sample and contacts before first lytic reagent.

34. the method for claim 29, wherein feed compartment comprises that first lytic reagent and sample contact first lytic reagent and occur in when sample placed feed compartment.

35. the method for claim 29 wherein places sample feed compartment to occur in sample and contacts after first lytic reagent.

36. the method for claim 29 wherein forms the sample concentration district and comprises and carry out centrifugal to the sample in the treatment chamber in the valve treatment chamber is arranged.

37. the method for claim 29, wherein solid phase material comprises the tetrafluoroethylene fibril-matrix, is trapped in the adsorptivity particle in the matrix and is applied to coating reagent on the solid phase material, and wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration.

38. the method for claim 29, wherein first lytic reagent is a nonionic surface active agent.

39. the method for claim 29 is wherein separated and is comprised the material that contains nucleic acid with eluent from solid phase material wash-out at least a portion.

40. be used for from the test kit of sample separation nucleic acid, this test kit comprises:

First lytic reagent;

Solid phase material, this solid phase material comprise the tetrafluoroethylene fibril-matrix, be trapped in the adsorptivity particle in the matrix and be applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; With

Teachings is used for according to the method for claim 2 sample dissociation being separated with solid phase material with the material and/or the nucleic acid that make at least a portion contain nucleic acid.

41. be used for from the test kit of sample separation nucleic acid, this test kit comprises:

First lytic reagent;

Solid phase material, this solid phase material comprise the tetrafluoroethylene fibril-matrix, be trapped in the adsorptivity particle in the matrix and be applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; With

Teachings is used for according to the method for claim 24 sample dissociation being separated with solid phase material with the material and/or the nucleic acid that make at least a portion contain nucleic acid.

42. be used for from the test kit of sample separation nucleic acid, this test kit comprises:

Nonionic surface active agent;

Solid phase material, this solid phase material comprise the tetrafluoroethylene fibril-matrix, be trapped in the adsorptivity particle in the matrix and be applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; With

Teachings is used for according to the method for claim 3 sample dissociation being separated with solid phase material with the material and/or the nucleic acid that make at least a portion contain nucleic acid.

43. be used for from the test kit of sample separation nucleic acid, this test kit comprises:

Nonionic surface active agent;

Solid phase material, this solid phase material comprise the tetrafluoroethylene fibril-matrix, be trapped in the adsorptivity particle in the matrix and be applied to coating reagent on the solid phase material; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; With

Teachings is used for according to the method for claim 25 sample dissociation being separated with solid phase material with the material and/or the nucleic acid that make at least a portion contain nucleic acid.

44. be used for from the test kit of sample separation nucleic acid, this test kit comprises:

First lytic reagent;

Microfluidic device, this microfluidic device comprise feed compartment, the valve treatment chamber is arranged and comprise the separate chamber of solid phase material; Wherein solid phase material comprises and catches the position, is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; With

Teachings is used for according to the method for claim 5 sample dissociation being separated with solid phase material with the material and/or the nucleic acid that make at least a portion contain nucleic acid.

45. be used for from the test kit of sample separation nucleic acid, this test kit comprises:

First lytic reagent;

Microfluidic device, this microfluidic device comprise feed compartment, the valve treatment chamber is arranged and comprise the separate chamber of solid phase material; Wherein solid phase material comprises and catches the position, is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; With

Teachings is used for according to the method for claim 28 sample dissociation being separated with solid phase material with the material and/or the nucleic acid that make at least a portion contain nucleic acid.

46. be used for from the test kit of sample separation nucleic acid, this test kit comprises:

Nonionic surface active agent;

Microfluidic device, this microfluidic device comprise feed compartment, the valve treatment chamber is arranged and comprise the separate chamber of solid phase material; Wherein solid phase material comprises and catches the position, is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; With

Teachings is used for according to the method for claim 10 sample dissociation being separated with solid phase material with the material and/or the nucleic acid that make at least a portion contain nucleic acid.

47. be used for from the test kit of sample separation nucleic acid, this test kit comprises:

Nonionic surface active agent;

Microfluidic device, this microfluidic device comprise feed compartment, the valve treatment chamber is arranged and comprise the separate chamber of solid phase material; Wherein solid phase material comprises and catches the position, is applied to coating reagent on the solid phase material or both; Wherein coating reagent is selected from the polymkeric substance barrier and the combination thereof of tensio-active agent, highly basic, polyelectrolyte, alternative infiltration; With

Teachings is used for according to the method for claim 29 sample dissociation being separated with solid phase material with the material and/or the nucleic acid that make at least a portion contain nucleic acid.